Identification and validation of novel targets for agrochemicals

ABSTRACT

The invention relates to a method for identifying and validating plant targets for agrochemicals, comprising the steps of determining gene or protein expression profiles in function of the progression of an essential biological process in a plant, and the subsequent downregulation of expression of said gene or protein in a plant cell. More particularly, the effects of downregulation of the candidate target gene were directly monitored on plants locally infected with a vector mediating viral induced gene suppression in that infected plant area. The invention also relates to isolated plant genes encoding proteins involved in plant growth and development. The invention also relates to plants tolerant to agrochemicals such as herbicides or pesticides.

The invention relates to isolated plant genes encoding proteinsessential for plant growth and development and to methods foridentifying and validating these genes/proteins as target genes/proteinsfor agrochemicals, such as herbicides. A target for an agrochemical is agene or a protein where the agrochemical interferes with when applied tothe target organism.

For the identification and validation of useful agrochemicals, theagrochemical industry traditionally relied on in vivo screening methodswherein chemical compounds were brought into direct contact with theliving target organisms (e.g. plants for herbicide screening, insectsfor insecticide screening, etc.). However due to (i) the dramaticincrease in the number of compounds that need to be screened to find asuccessful new agrochemical product, and (ii) the need to rely on verysmall quantities of compound such as are available in a combinatorialchemistry based compound libraries, and (iii) the need to identifycompounds with a novel mode of action, the industry has developed aconsiderable interest in using more efficient and faster in vitroscreening methods.

To render such in vitro screening methods more successful, it isessential to carefully select the tested target gene/proteins and/or thetested agrochemicals. It has been described that a more practical invitro approach for finding new agrochemicals would involveidentification of target genes/proteins against which the agrochemicalcompounds could possibly work. For this process identification ofsuitable target genes/proteins, the conventional methods make use ofgene knock-outs of the target organism. Gene knock-out libraries aregenerally made as a random collection of thousands of gene knock-outs.In these methods it is investigated if the gene/protein is essential forthe growth and/or viability of the organism, since the knockout of anessential gene (when present in a homozygous state) leads to a lethal orotherwise detrimental effect on the organism. The indication that saidgene/protein is essential to the organisms makes it a suitable targetfor an agrochemical. These conventional methods are still cumbersome andtime consuming because of the use of gene-knockouts. Other techniquesthat are useful to estimate the essential character of a gene or its.corresponding protein are based on the downregulation of said gene orprotein for example via anti-sense expression technology (WO01/07601).

To render an in vitro screening for agrochemicals more successful, it isessential to carefully select the tested target gene/proteins. Thereforea more practical in vitro approach for finding new agrochemicals couldbe a multistep process involving the steps of (1) identification oftarget genes/proteins against which the agrochemical compounds couldpossibly work, (2) validation of the candidate target gene as being anessential gene/protein for the organism and (3) use of these targetgenes/proteins in an in vitro screening procedure in which the chemicalcompounds are tested.

It is the aim of the present invention to develop a process for the moreefficient identification of candidate target genes/proteins foragrochemicals, combined with the more efficient validation of the targetgenes/proteins. It is a further aim of the invention to provide thisprocess in order to design more efficiently the screening procedure withthe agrochemical compound.

The method of the present invention is based on the direct use ofgenetic information for example generated by expression profiling of thecandidate target -genes/proteins, for the identification and thevalidation of the targets.

Therefore according to a first embodiment of the present invention,there is now provided a method for identifying and validating plantgenes/proteins as targets for agrochemicals, said method comprising thesteps of:

-   -   a. determining gene or protein expression profiles during a        biological process of a plant or plant cell, said biological        process being necessary for the viability or the growth of the        plant or plant cell;    -   b. selecting genes or proteins having altered expression during        said biological process,    -   c. cloning said selected gene or the nucleic acid encoding said        protein in its full-length or partial form,    -   d. incorporating said nucleic acid in a vector designed for        downregulation of expression of said nucleic acid or the        sequence homologous to said nucleic acid in a plant or plant        cell.

The aim of methods of the present invention is the identification oftarget gene(s)/protein(s) out of a broad range of candidate plantgenes/proteins. The identification step is achieved by the techniques ofexpression profiling described in the following embodiments. Since themethod of the present invention can be used for identification ofgenes/proteins or proteins, the term “target” as used herein can mean agene as well as a gene product, namely a protein, polypeptide orpeptide. With the expression “target for an agrochemical” is meant aprotein as well as a gene or nucleic acid encoding such protein, andwhen such target is inhibited, stimulated or otherwise disrupted in itsnormal activity by an agrochemical compound, this would lead to adesired effect in a target organism. The invention aims at efficientlyidentifying targets for agrochemicals. Said agrochemicals can beherbicides or pesticides as well as growth stimulators or growthregulators.

Target identification means selecting candidate targets from a largernumber of genes/proteins or proteins on the basis of certain propertiesthat give such a molecule a higher probability of being a suitabletarget than other molecules which do not exhibit said properties. Aherbicide target is a protein or gene that when inhibited, stimulated orotherwise disrupted in its normal activity by a compound would kill the(weedy) target plant or have a strong negative effect on its growth,said compound would therefore be a candidate herbicide. An insecticidetarget is a protein or gene that when inhibited, stimulated or otherwisedisrupted in its normal activity by a compound would kill the insectpest or have a strong negative effect on its growth, said compound wouldtherefore be a candidate insecticide. A plant growth regulator (PGR)target is a protein or gene that when inhibited, stimulated or otherwisedisrupted in its normal activity by a compound would promote or alter ina desirable way the growth of plant, said compound would therefore be acandidate PGR.

Nowadays a lot of genomic information, e.g. gene sequences, expressionprofiles, homologies and putative functionality, is available fromgenomic sequencing and expression studies in several target organisms.It is therefore of interest to develop a new method to identify andvalidate genes/proteins as candidate targets for agrochemicals, suchmethods being based on a direct use of such genomic information. Thisuse of genomic information, e.g. the expression level of a gene, allowsthe selection of a limited set of appropriate candidate genes/proteins.Only this limited set of genes is then tested in the validation step,contributing to a higher efficiency and success rate of the screeningprocedure for agrochemicals. Furthermore, the genetic information, e.g.the functional data of the putative target gene/protein, is used as abasis to design more efficiently the in vitro screening procedure withthe agrochemical compound(s) under investigation.

The present invention discloses methods that allow for theidentification and validation of target genes/proteins for agrochemicalsout of the broad range of possible genes/proteins and proteins. Ittherefore allows genes or proteins to be selected for the development ofsuitable in vitro screening methods for the screening of novel andefficient agrochemicals.

According to a first step of the methods of the present invention targetgenes or gene products are identified by using transcript profiling ofthe genomic content of a cell. By using this technique one immediatelyobtains genomic data (sequences and expression level) as well as afunctional indication of the candidate target gene or gene product. Thusthis method is useful for a first identification and selection ofpossible agrochemical target genes/proteins, since it provides as abonus genomic and functional data on the candidate target. A goodcandidate target gene is a gene of which the expression variessignificantly over the course of an essential biological process of thecell, since that is an indication that the gene/protein is involved inthat biological process The present application describes for the firsttime that the determination of an expression profile of a gene duringthe progression of an essential biological process is used to identifypossible agrochemical targets.

The expression profiling in the target identification steps of themethod of the present invention is carried out in function of theprogression of a process that is essential for plant growth and/or plantdevelopment and/or plant viability. In one preferred embodiment of thepresent invention, the essential process that is monitored in the targetidentification step is the process of cell division. Accordingly, in aparticular embodiment of the invention, the method to identify targetgenes/proteins for agrochemicals is based on the transcript profiling ofgenes/proteins that are specifically involved in cell division.Therefore the invention provides a method as mentioned above, whereinsaid biological process cell division.

Other biological processes that may be monitored for the identificationand validation of agrochemical targets are for instance processes thatare essential for seed germination, leaf formation, etc.

The term expression profiling means determining the time and/or placewhen or where a gene or a protein is active. Particularly for a gene,this is achieved by monitoring the level of transcripts and therefore inthe case of gene expression profiling the term transcript profiling ormRNA profiling is used.

Generally, the expression profiling in the methods of the presentinvention is carried out in function of the progression of a processthat is essential for plant growth and/or development and/or plantviability. To achieve this, the process of interest is synchronized in asufficient number of cells (for example in a cell culture) or organismsto allow collecting samples for expression profiling representingvarious stages of said process. Target identification then consists inselecting those genes or proteins that show significant changes inexpression levels in function of the progression of the process ofinterest. It are those genes or proteins that are likely to be stronglyinvolved or to be essential in said process.

The term “essential” means that if the gene or the gene product cannotfunction as normal in the cell or organism, this will have significantimplication in the cell growth or cell development or other vitalfunctions of the cell or organism.

According to the invention, the expression profiling can be studied atthe level of m-RNA, using transcript profiling techniques, oralternatively at the level of protein, using proteomics-basedapproaches.

In one preferred embodiment of the invention, m-RNA profiling is usedfor identification of target genes/proteins and expression levels may bequantified via techniques that are well known to the man skilled in theart. For instance, mRNA-profiling can be performed using micro-array ormacro-array technologies, this method however requires that the genesequences are known (full length sequences or at least partialsequences) and are physically available for coating on the micro ormacro array surface. Standard chips are being commercialised forArabidopsis, and sufficient sequence information is now available fordifferent plant species (including rice) to allow sufficient sequencedata for this approach. Another approach for mRNA profiling is the useof AFLP-based transcript profiling as described in example 1. In thisapproach short sequence tags are monitored. In a next step these shortsequence tags may be matched with full-length genes/proteins ifrequired. Gene or protein selection thus be based on either full-lengthor partial sequences and it is well within the realm of the personskilled in the art to find a full length sequence based on the knowledgeof a partial sequence.

Therefore, one aspect of the invention is the direct use of geneticinformation to select candidate targets for agrochemicals. As mentionedabove this genetic information can be generated by a number oftechniques. Accordingly, the present invention encompasses a method asmentioned above, wherein the expression profiles are determined by meansof micro-array, macro array or c-DNA-AFLP.

According to another embodiment of the invention, proteomic basedapproaches may be used to identify candidate target proteins foragrochemicals.

It is now demonstrated that for the purposes of identifying a targetgene for agrochemicals a synchronized culture of dividing plant cells isused to isolate samples and to monitor the expression of the transcriptsof those cells during the progression of the cell division.

Therefore according to a particular embodiment, the invention alsoencompasses a method for the identification and validation of plantagrochemical targets, wherein said gene or protein expression profilingis based on nucleic acid or protein samples collected from asynchronized culture of dividing plant cells.

In one embodiment of the invention, the samples used for expressionprofiling are obtained from a synchronized culture of rice cells,tobacco cells, Arabidopsis cells or cells from any other plant species.The cell culture should be synchronized in order to obtain samplescontaining a sufficient amount of cells that are at the same stage ofthe biological process, so that the various samples taken for expressionprofiling are representative for the various stages of the essentialbiological process. In a particular embodiment of the present inventionthe samples are obtained from cells that are synchronized for celldivision. In a preferred embodiment of the invention expressionprofiling is done on synchronized dividing cells. Certain cell lines areparticularly suitable for synchronization of cell division, for instancesynchronization of tobacco Bright Yellow-2 cell lines as described inexample 1. Therefore most preferably, the synchronized cells are tobaccoBY2 cells. By using synchronized tobacco BY2 cells and performing acDNA-AFLP-based genome-wide expression analysis, the inventors built alarge collection of plant cell cycle-modulated genes/proteins.Approximately 1340 periodically expressed genes/proteins wereidentified, including known cell cycle control genes as well as numerousnovel genes. A number of plant-specific genes were found for the firsttime to be cell cycle modulated. Other transcript tags were derived fromunknown plant genes showing homology to cell cycle-regulatory genes ofother organisms. Many of the genes encode novel or uncharacterisedproteins, indicating that several processes underlying cell division arestill largely unknown. These sequences are presented herein as SEQ ID NO1 to SEQ ID NO 785.

While, according to the invention, the basic criterion for identifyingan agrochemical target gene or gene product consists in the differentialexpression levels of the gene or the protein observed during theprogression of an essential biological progress, secondary selectioncriteria can be used and combined with this primary criterion.

One such secondary criterion may be to make a selection of genes orproteins that are found not to exhibit a high degree of homology withgenes or proteins from other organisms (such as mammals) as thiscriterion is likely to reduce the probability that the agrochemicalcompounds active on the “plant-specific” target genes or gene productswould also exhibit toxic effects against other organisms, for examplemammals.

Another secondary selection criterion could exist in focussing on aparticular phase of the essential biological process as mentioned above.For instance, when cell division modulated genes/proteins are underinvestigation as potential agrochemical target genes/proteins, one couldpreferably use those cell division modulated genes/proteins whichexhibit high expression during the G1 phase, S phase, G2 phase or Mphase or at the transition stages of these phases. In one embodiment ofthe present invention, the focus may be on the G2/M transition phase,since this phase in the plant cell cycle is considered to have more“plant specific” elements than other phases of the cell cycle and istherefore more likely to yield plant specific candidate target genes andproteins. Whereas the core cell cycle genes/proteins and the basicregulatory mechanisms controlling cell cycle progression are conservedamong higher eukaryotes, basic developmental differences between plantsand other organisms imply that plant-specific regulatory pathways existthat control cell division. Especially for events occurring at mitosis,plants are expected to have developed unique mechanisms regulatingkaryo- and cytokinesis. A typical plant cell is surrounded by a rigidwall and can as such not divide by constriction. Instead, a new cellwall between daughter nuclei is formed by a unique cytoskeletalstructure called the phragmoplast, whose position is dictated by anothercytoskeletal array called the preprophase band. Another major differencebetween plant and animal mitosis is found in the structure of themitotic spindles: in animals, they are tightly centred at thecentrosome, whereas in plants they have a diffuse appearance.

Therefore a suitable second criterion to combine with the firstcriterion may be to select genes/proteins that are involved in themitosis step of the cell cycle and/or that are involved in the buildingof the cell wall during mitosis.

Likewise a secondary selection criterion to be combined with the firstcriterion may be the selection of genes or proteins from adicotyledonous plant that do not exhibit a high degree of homology withgenes or proteins from a monocotyledonous plant (or vice versa). Thissecondary criterion is especially relevant when identifying agrochemicaltarget genes or proteins with the intention to selectively identifytargets that would allow for subsequence screening of selectiveherbicides or plant growth regulators. For instance, this strategy isadvantageous to find targets and agrochemicals for selective weedcontrol, such as herbicides that kill dicotyledonous weeds inmonocotyledonous crops or vice versa.

Therefore according to further embodiments, the present inventionencompasses methods as mentioned above, wherein the target gene orprotein meets any one or more of the above mentioned secondary selectioncriteria, such as being plant specific, being mitosis specific or beingdicot specific etc.

The possibility for combination of criteria used for selecting targetgenes or proteins renders the method of the present invention morepowerful than classical methods. According to a preferred embodiment thetechnique of the present invention allows identifying genes/proteins, tobe used as agrochemical target genes/proteins, these genes beinggenes/proteins that are involved in cell division and control of cellcycle progression, and these genes being novel and these genes beingplant specific. Therefore the method of the present invention ischaracterized in that it allows identifying new and unexpectedagrochemical targets.

In the target gene identification step according to the presentinvention, genes or proteins are selected for which there is a highprobability of being essential. It should be clear that theabove-mentioned examples are given by way of illustration and are notmeant to be limiting in any way.

Further, according to a second step in the method of the invention, thecandidate agrochemical target gene or gene product is subsequentlyvalidated as being essential for the growth and/or development and/orviability of the organism. This is achieved by cloning the identifiedcandidate target gene in a vector construct designed to downregulatesaid target gene in a plant or plant cell, followed by inoculating theplant with this construct and monitoring whether downregulation of thegene results in negative effects on plant growth and/or developmentand/or viability. A valid target gene is a target gene that causessignificant effects on growth of plants or plant cells whendownregulated. The present application describes for the first time theuse of a particularly fast and efficient downregulation method tovalidate possible agrochemical targets.

Accordingly, the present invention encompasses a method as mentionedabove for the identification and validation of plant targets foragrochemicals, wherein said downregulation involves a viral-induced genesilencing mechanism.

Thus, starting from a number of candidate target genes/proteinsidentified in the first step of the method of the invention, the targetvalidation step aims at confirming and demonstrating the essentialnature of the gene by demonstrating that severe down-regulation of theexpression level of the gene has a significant effect on the organism.

In particular, when one is interested in developing a screening assayfor herbicides, downregulation of the candidate target gene in a plantmay result in a lethal effect, a severe inhibition of plant growth orany other (obviously) negative phenotypic effects. Alternatively, whenone is interested in developing a screening assay for plant growthregulators, the effect of downregulating the target gene may bemodulation or even stimulation of growth in general or modulation oreven stimulation of a particular process associated with plant growthand/or development and/or architecture and/or physiology and/orbiochemistry or any other phenotypic effect.

The man skilled in the art will be aware of various methods to achievedownregulation of a given gene or protein, such methods includeessentially co-suppression based approaches or anti-sense basedapproaches as well as any other method resulting in gene silencing.Other examples of downregulation in a cell are well documented in theart and include, for example, RNAi techniques, the use of ribozymes etc.Gene silencing may also be achieved by insertion mutagenesis (forexample, T-DNA insertion or transposon insertion) or by gene silencingstrategies as described by, among others, Angell and Baulcombe, 1998 (WO98/36083), Lowe et al., 1989 (WO 98/53083), Lederer et al., 1999 (WO99/15682) or Wang et al., 1999 (WO 99/53050). Expression of anendogenous gene may also be reduced if the endogenous gene contains amutation.

The effect of gene downregulation can be observed in stably transformedplants which can be obtained by means of various well known techniques,these techniques generally involving a plant transformation step and aplant regeneration step.

Genes/proteins which exhibit a severe negative effect when downregulatedmay however significantly reduce transformation and/or regenerationefficiency. Therefore, a relevant parameter indicative for the essentialnature of the gene, may be a severe reduction in transformationefficiency when said particular gene is used in a down-regulationconstruct. In order to avoid the (negative) effect on transformationefficiency in the transformation and regeneration process, an induciblepromoter system can be used. Induction of promoter activity can then beapplied at a later stage (after transformation) in order to observe theeffect of gene downregulation once the transformed plant or plantletstarted to develop.

Further, another method for testing the effect of downregulation of atarget gene, which can be used in the methods of the present invention,is based on a rapid transient transformation process and does not relyon the somewhat lengthy process of stable transformation. The use ofthis method for target validation in plants is part of this invention,regardless of whether target identification has been performed accordingto this invention.

Accordingly, in a preferred embodiment, the downregulation method isbased on co-suppression and on rapid transient transfection of plantcells. The preferred method to validate genes/proteins as targets foragrochemicals is based on the cloning of the identified candidate targetgene in a vector construct containing a viral replicase that is involvedin the very efficient downregulation of the candidate target gene in theinfected plant or plant cell via the mechanism of co-suppression. Oneadvantage of this method for downregulation, is the fact that theinfection of the host cells or the plant can be performed locally forexample by inoculating the vector directly on the leaves. This allows avery fast evaluation of the effect of downregulating the candidatetarget since no complete transgenic plants have to be generated. Alsothis technique allows an easy way of monitoring the effect of thedownregulated candidate target by simply looking at the changes of theinfected place, for example monitoring the lethal effects on theinfected leaf.

Therefore in a preferred embodiment, the downregulation method is basedon co-suppression. In a more preferred embodiment of the invention thisco-suppression technique is fast and easy to evaluate the effect ofdownregulation, so that it is suitable for dealing with high numbers ofgenes/proteins. This can be achieved by using viral induces genesilencing mechanisms (VIGS) and by infecting the plant directly andlocally, for example on the leaves. Therefore, according to anotherembodiment, the present invention relates to the use of a viral-inducedgene silencing system for validating plant targets for agrochemicals.

This method for severe downregulation via transient expression of thegene in the presence of certain viral elements is referred to as“virus-induced gene silencing mechanism” (VIGS) and is previouslydescribed in Ratcliff et al., Plant J., 25 237-245, 2001. Briefly, virusvectors carrying host-derived sequence inserts induce silencing of thecorresponding genes/proteins in infected plants. This virus-induced genesilencing is a manifestation of an RNA-mediated defence mechanism thatis related to post-transcriptional gene silencing in transgenic plants.Ratcliff et al, developed an infectious cDNA clone of Tobacco rattlevirus (TRV) that has been modified to facilitate insertion of non-viralsequences and subsequent infection in plants. This vector mediates VIGSof endogenous genes/proteins in the absence of virus-induced symptoms.Unlike the other RNA virus vectors that have been used previously forVIGS, the TRV construct is able to target most RNA's in the growingpoints of the plant. A more detailed description of this downregulationmechanism is given in example 2.

According to particular embodiments of the present invention, the VIGSsystem is applied in Arabidopsis or in tobacco for the purposes ofvalidation of a candidate agrochemical target gene.

According to a further preferred embodiment, there is provided a methodfor validation of a candidate agrochemical target gene, wherein the geneis downregulated in a plant via the use of infectious DNA of virus isTobacco Rattle Virus and wherein said plant is tobacco.

The present invention relates to a combination of the above-mentionedidentification and validation steps, which are especially selected sothat they lead to an efficient selection of candidate target genes foragrochemicals. The outcome of the transcript profiling provides thenecessary information and forms the basis for the second step, namelythe validation of the target gene via incorporation of the gene sequencein the downregulation construct. The combination of these two techniquesis especially useful for selecting suitable target genes/proteins foragrochemicals in a high throughput fashion. This technique thusovercomes the technical limitations of previously described techniquessuch as the knock-out libraries and the antisense strategies withoutgenetic information of the genes. This new combination offers atime-saving strategy for identification of a candidate target gene andthe more direct information output in the form of a real sequence, theimmediate cloning of the gene in the downregulation construct andimmediate application of the downregulating construct on the targetorganism.

The combination of these steps offers the unique opportunity to providemany high quality target genes/proteins for agrochemicals in acommercially and economically advantageous way. Furthermore, inherent tothe techniques of the present invention is that the qualified targetgenes/proteins are accompanied with the necessary information to designa suitable in vitro screening assay with the agrochemical. Thisinformation consists of the expression characteristics of thegenes/proteins and their function and importance in the essentialbiological process that was monitored during the transcript profiling.

In this way, the methods of the present invention overcome the practicaland commercial limitations of the existing techniques.

Once this level of target validation is reached, the validated targetcan be selected for the development of an appropriate high-throughput invitro screening method, wherein the agrochemical is tested. Therefore,the present invention also encompasses a method for screening candidateagrochemical compounds, comprising the use of any of the identificationprocedures and/or validation procedures as mentioned above. Moreparticularly, the present invention encompasses a method for screeningagrochemical compounds, comprising the use of any one or more of thesequences represented in SEQ ID NO 1 to 785.

Various methods can be used to develop suitable in vitro assays forscreening the chemical compounds, depending on what is known about thebiological activity of the target gene. For example, when the target isan enzyme, measurement of the enzymatic activity of the target couldform the basis of the in vitro screening assay with the chemicalcompound.

Therefore, the methods of the present invention, the genes/proteins andthe information generated by the combined identification and validationmethods of the present invention, allow one to design and/or fine tune ascreening for testing and/or developing agrochemicals (for exampleherbicides). For example if the expression pattern and the role of thetarget gene in the essential biological process is known, it is mucheasier to set up an in vitro screening assay to monitor the effect of acandidate herbicide on the target cells. Therefore it is expected thatmuch more refined and/or efficient herbicides will be characterizedusing the methods of the present invention.

Also because of the knowledge of its function, one can further designthe screened agrochemical compound to improve its activity for instanceto improve its binding capacity to the target.

Therefore, the present invention encompasses a method for screeningcandidate agrochemical compounds comprising the use of any of themethods as mentioned above.

The invention may also be applied for the development of agrochemical(for example herbicide or pesticide) tolerant plants, plant tissues,plant seeds and plant cells.

Herbicides that exhibit greater potency can also have greater cropphytotoxicity. A solution to this problem is to develop crops that areresistant or tolerant to herbicides. Crop hybrids or varieties that aretolerant to the herbicides allow, for instance, for the use ofherbicides that kill weeds without attendant risk of damaging the crop.Further it should be clear that when a plant is overexpressing thetarget of a particular herbicide, the tolerance of said plant againstsaid herbicide will also be enhanced.

Therefore the present invention also relates to the use of theagrochemical (e.g. herbicide) target genes/proteins as identified by themethod of the present invention for generating transgenic plants thatare tolerant or resistant to an agrochemical (e.g. herbicide). Exampleof genes and gene sequences identified by the combined identificationand validation methods of the present invention and which can be used asagrochemical target or that can be used to obtain herbicide tolerantplants comprise the sequences as represented in any of SEQ ID NOs 1 to785.

These sequences are derived from tobacco, but the one skilled in the artcan easily find via homology search in databases or homology search in acDNA library the homologues genes of other plant species, for instancemonocot sequences (e.g. the corresponding rice or corn sequence), anduse them for the same purposes as described herein. These homologysearches can be done for example with a BLAST program (Altschul et al.,Nucl. Acids Res., 25 3389-3402, 1997) on a sequence database such as theGenBank database. Homology studies as referred to above can be performedusing sequences present in public and/or proprietary databases and usingseveral bioinformatics algorithms, well known to the man skilled in theart. Methods for the alignment of sequences are well known in the art,such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses thealgorithm of Needleman and Wunsch (J. Mol. Biol. 48: 443-453, 1970) tofind the alignment of two complete sequences that maximizes the numberof matches and minimizes the number of gaps. The BLAST algorithmcalculates percent sequence identity and performs a statistical analysisof the similarity between the two sequences. The software for performingBLAST analysis is publicly available through the National Centre forBiotechnology Information.

Further, some of the tobacco sequences identified by the method of thepresent invention might be partial but again, the full-length sequencecan easily be found based on the partial sequence. For example“transcript building” can be done based on homology search on ESTdatabases, cDNA's or gene predictions. These databases and programs arepublicly available e.g. http://www.tigr.org/.

Therefore the present invention relates to the use of the nucleic acidsas identified and disclosed herein and represented in SEQ ID NO 1 to785, and also to the use of the full length genes regenerated from thepartial sequences as well as to the use of the homologues sequencesisolated from the same or from other plants.

In another embodiment, the present invention relates to a nucleic acididentified according to the method of the invention. Thus the inventionencompasses an isolated nucleic acid identifiable by any of the methodsas mentioned above.

In another embodiment, the invention relates to a nucleic acididentified according to the method of the invention, comprising thenucleic acid sequence chosen from the group of SEQ ID NO 1 to 785 or afull length sequence thereof, or a functional homologue thereof, or afunctional fragment thereof, or an immunologically active fragmentthereof. Thus the invention encompasses an isolated nucleic acid,comprising at least part of a nucleic acid sequence chosen from thegroup of SEQ ID NO 1 to 785 a homologue, functional fragment orderivative thereof.

With “a functional fragment” is meant any part of the sequence that isresponsible for the biological function or for an aspect of thebiological function of the nucleic acid sequence.

Further, the invention encompasses a method for the production of anagrochemical resistant plant, comprising the use of any one or more ofSEQ ID NO 1 to 785 or a homologue, functional fragment or derivativethereof or one or more of the proteins encoded by SEQ ID NO 1 to 785 ora homologue, functional fragment or derivative thereof.

In one embodiment of the present invention the sequences, thefull-length sequences and the homologues are used to develop herbicidetolerant plants.

Further the invention encompasses a plant tolerant to an agrochemical,in which the expression level of one or more of the nucleic acidscorresponding the SEQ ID NO 1 to 785 or the homologue, functionalfragment or derivative thereof, is modulated. Further the inventionencompasses any part or more preferably any harvestable part of theseplants.

Therefore the invention also relates to the use of these sequences, thefull-length sequences and the homologues as targets for agrochemicalsThe invention encompasses the use of a nucleic acid as mentioned aboveor the protein encoded by said isolated nucleic acid as a target for anagrochemical compound, preferably, wherein the agrochemical compound isa herbicide.

Further, the invention relates to the use of these sequences to developscreening assays for the identification and/or development ofagrochemicals. The invention encompasses a method for screeningcandidate agrochemical compounds comprising the use of any one or moreof SEQ ID NO 1 to 785 or a homologue, functional fragment or derivativethereof or one or more of the proteins corresponding to SEQ ID NO 1 to785 or a homologue, functional fragment or derivative thereof.

The present invention will be further illustrated by the followingfigures, wherein,

FIG. 1 shows the gene expression profiles obtained by quality-basedclustering of all transcript tags monitored in a transcript profilingexperiment as described in example 1. Shown are the trend lines of 16clusters containing 97% of the genes and covering the entire time courseas indicated on top. S-phase-specific gene clusters are grouped in A,gene clusters with peak expression between S- and M-phase are grouped inB, whereas group C contains the M- and G1-phase-specific clusters. D:Three small clusters of genes with peak expression during two cell cyclephases.

FIG. 2 shows the phenotypes of tobacco plants inoculated with aacetolactate synthase (SEQ ID NO 18) downregulation construct andphenotypes of tobacco plants inoculated with a prohibitin (SEQ ID NO 21)downregulation construct. The phenotypes were observed 12 days afterinoculation (upper panel) or 17 days after inoculation (lower panel).

FIG. 3 shows the phenotype of tobacco plants inoculated with a B-typeCDK (SEQ ID NO 11) downregulation construct. The observations were made37 days after inoculation.

FIG. 4 shows the sequences identified by the methods of the presentinvention and represented by SEQ ID NO 1 to SEQ ID NO 785

EXAMPLES Example 1

A cDNA-AFLP based expression profiling of sequence obtained from samplesof a synchronized tobacco BY2 cell line system, was used to identifygenes that are upregulated during the cell cycle, an essentialbiological process needed for the viability and growth of the tobaccocell line system.

A genome-wide expression analysis of cell cycle-modulated genes in thetobacco Bright Yellow-2 (BY2) cell line was performed. This unique cellline can be synchronized to high levels with different types ofinhibitors of cell cycle progression (Nagata et al., Int. Rev. Cytol.,132 1-30, 1992; Planchais et al., FEBS Lett., 476 78-83, 2000). Becauseof the lack of extensive molecular resources such as genomic sequences,cDNA clones or expressed sequence tags (ESTs) for tobacco, amicroarray-based approach cannot be used for a transcriptome analysis.Therefore, the cDNA-AFLP technology was used to identify andcharacterize cell cycle-modulated genes in BY2. cDNA-AFLP is a sensitiveand reproducible fragment-based technology that has a number ofadvantages over other methods for genome-wide expression analysis(Breyne and Zabeau, Curr. Opin. Plant Biol., 4 136-142, 2001): it doesnot require prior sequence information, it allows identification ofnovel genes, and it provides quantitative expression profiles. After adetailed analysis, it was found that around 10% of the transcriptsanalyzed is periodically expressed. This comprehensive collection ofplant cell cycle-modulated genes provides a basis for selecting andvalidating novel and unexpected agrochemical target genes

Synchronization of BY2 Cells and Sampling of Material.

Tobacco BY2 (Nicotiana tabacum L. cv. Bright Yellow-2) cultured cellsuspension were synchronized by blocking cells in early S-phase withaphidicolin as follows. Cultured cell suspension of Nicotiana tabacum L.cv. Bright Yellow 2 were maintained as described (Nagata et at., Int.Rev. Cytol., 132 1-30, 1992). For synchronization, a 7-day-oldstationary culture was diluted 10-fold in fresh medium supplemented withaphidicolin (Sigma-Aldrich, St. Louis, Mo.; 5 mg/l), a DNA-polymerase ainhibiting drug. After 24 h, cells were released from the block byseveral washings with fresh medium and resumed their cell cycleprogression. After the drug had been washed, samples were taken everyhour, starting from the release from the aphidicolin block (time 0)until 11 h later. The mitotic index was determined by counting thenumber of cells undergoing mitosis under fluorescence microscopy afterthe DNA had been stained with 5 mg/l 4′,6-diamidino-2-phenylindole(Sigma-Aldrich). DNA content was measured by flow cytometry. This wasdone as follows A subsample was used to check cell cycle progression andsynchrony levels. After the DNA had been stained with 5 mg/l4′,6-diamidino-2-phenylindole (Sigma-Aldrich), the mitotic index wasdetermined under fluorescence microscopy by counting the number of cellsundergoing mitosis. A mitotic peak of approximately 40% was obtained 8 hafter washing. For flow cytometry, cells were first incubated in abuffered enzyme solution (2% cellulase and 0.1% pectolyase in 0.66 Msorbitol) for 20 min at 37° C. After the suspension had been washed andresuspended in Galbraith buffer (Galbraith et al., Science, 2201049-1051, 1983), it was filtered through a 30-μm nylon mesh to purifythe DAPI-stained nuclei. The fluorescence intensity was measured using aBRYTE HS flow cytometer (Bio-Rad, Hercules, Calif.). Exit from S-phasewas observed 4 h after aphidicolin release and the level of synchronywas shown to be sufficiently high throughout the time course.

RNA Extraction and cDNA Synthesis.

Total RNA was prepared by using LiCl precipitation (Sambrook et al.,1989) and poly(A⁺) RNA was extracted from 500 μg of total RNA usingOligotex columns (Qiagen, Hilden, Germany) according to themanufacturer's instructions. Starting from 1 μg of poly(A⁺) RNA,first-strand cDNA was synthesized by reverse transcription with abiotinylated oligo-dT₂₅ primer (Genset, Paris, France) and SuperscriptII (Life Technologies, Gaithersburg, Md.). Second-strand synthesis wasdone by strand displacement with Escherichia coli ligase (LifeTechnologies), DNA polymerase I (USB, Cleveland, Ohio) and RNAse-H(USB).

cDNA-AFLP Analysis.

Five hundred ng of double-stranded cDNA was used for AFLP analysis asdescribed (Vos et al., Nucl. Acids Res., 23 4407-4414, 1995; Bachem etal., Plant J., 9 745- 753, 1996) with modifications. The restrictionenzymes used were BstYI and MseI (Biolabs) and the digestion was done intwo separate steps. After the first restriction digest with one of theenzymes, the 3′ end fragments were collected on Dyna beads (Dynal, Oslo,Norway) by means of their biotinylated tail, while the other fragmentswere washed away. After digestion with the second enzyme, the releasedrestriction fragments were collected and used as templates in thesubsequent AFLP steps. The adapters used were: for BstYI,5′-CTCGTAGACTGCGTAGT-3′ and 5′-GATCACTACGCAGTCTAC-3′, and for MseI,5′-GACGATGAGTCCTGAG-3′ and 5′-TACTCAGGACTCAT-3′; the primers for BstYIand MseI were 5′-GACTGCGTAGTGATC(T/C)N_(1,2)-3′ and5′-GATGAGTCCTGAGTAAN_(1,2)-3′, respectively. For preamplifications, aMseI primer without selective nucleotides was combined with a BstYIprimer containing either a T or a C as 3′ most nucleotide. PCRconditions were as described Vos et al., Nucl. Acids Res., 23 4407-4414,1995): The obtained amplification mixtures were diluted 600-fold and 5μl was used for selective amplifications using a P³³-labeled BstYIprimer and the Amplitaq-Gold polymerase (Roche Diagnostics, Brussels,Belgium). Amplification products were separated on 5% polyacrylamidegels using the Sequigel system (Biorad). Dried gels were exposed toKodak Biomax films as well as scanned in a phosphoImager (AmershamPharmacia Biotech, Little Chalfont, UK).

Quantitative measurements of the expression profiles and data analysis.Gel images were analyzed quantitatively with the AFLP-QuantarPro imageanalysis software (Keygene N.V., Wageningen, The Netherlands). Thissoftware was designed for accurate lane definition, fragment detection,and quantification of band intensities. All visible AFLP fragments werescored and individual band intensities were measured per lane. Theobtained data were used to determine the quantitative expression profileof each transcript. The raw data were corrected for differences in totallane intensities, after which each individual gene expression profilewas variance-normalized . This was done as follows.

The obtained raw data were first corrected for differences in total laneintensities which may occur due to loading errors or differences in theefficiency of PCR amplification with a given primer combination for oneor more time points. The correction factors were calculated based onconstant bands throughout the time course. For each primer combination,a minimum of 10 invariable bands was selected and the intensity valueswere summed per lane. Each of the summed values was divided by themaximal summed value to give the correction factors. Finally, all rawvalues generated by QuantarPro were divided by these correction factors.

Subsequently, each individual gene expression profile wasvariance-normalized by standard statistical approaches as used formicroarray-derived data (Tavazoie et al., Nature Genet., 22 281-285,1999). For each transcript, the mean expression value across the timecourse was subtracted from each individual data point after which theobtained value was divided by the standard deviation. A coefficient ofvariation (CV) was calculated by dividing the standard deviation by themean. This CV was used to establish a cut-off value and all expressionprofiles with a CV less than 0.25 were considered as constitutivethroughout the time course.

The Cluster and TreeView software (Eisen et al., PNAS, 95 14863-14868,1998) was used for hierarchical, average linkage clustering.Quality-based clustering was done with a newly developed softwareprogram (De Smet et al., Bioinformatics 2002 May; 18(5): 735-46). Thisprogram is related to K-means clustering, except that the number ofclusters does not need to be defined in advance and that the expressionprofiles that do not fit in any cluster are rejected. The minimal numberof tags in a cluster and the required probability of genes belonging toa cluster were set to 10 and 0.95, respectively. With these parameters,86% of all the tags were grouped in 21 distinct clusters.

Characterization of AFLP Fragments.

Bands corresponding to differentially expressed transcripts wereisolated from the gel and eluted DNA was reamplified under the sameconditions as for selective amplification. Sequence information wasobtained either by direct sequencing of the reamplified polymerase chainreaction product with the selective BstYI primer or after cloning thefragments in pGEM-T easy (Promega, Madison, Wis.) or sequencing ofindividual clones. The obtained sequences were compared againstnucleotide and protein sequences present in the publicly availabledatabases by BLAST sequence alignments (Altschul et al., Nucl. AcidsRes., 25 3389-3402, 1997). When available, tag sequences were replacedwith longer EST or isolated cDNA sequences to increase the chance offinding significant homology. Based on the homology, transcript tagswere classified in functional groups as shown in Table 1.

Experimental Results

Identification and Characterization of Cell Cycle-Modulated Genes

Tobacco BY2 cells were synchronized by blocking cells in early S-phasewith aphidicolin, an inhibitor of DNA polymerase α. After the inhibitorhad been released, 12 time points with an 1-h interval were sampled,covering the cell cycle from S-phase until M-to-G1 transition. Flowcytometry and determination of the mitotic index showed that themajority of cells exit S-phase 4 h after release from blocking and thatthe peak of mitosis is reached at 8 h. From each time point, extractedmRNA was subjected to cDNA-AFLP-based transcript profiling.

Quantitative temporal accumulation patterns of approximately 10,000transcript tags were determined and analyzed. In total, around 1,340transcript tags were modulated significantly during the cell cycle.Hierarchical clustering of the expression profiles resulted in fourlarge groups with the peak of expression in S-, early G2-, late G2-, orM-phase. Within each of these groups, several smaller clusters of geneswith similar expression patterns could be distinguished. Byquality-based clustering 21 different clusters were identified (see:http://www.plantgenetics/genomics/CCMgenes). In agreement with thehierarchical clustering, the four largest clusters (clusters 1 to 4 inFIG. 1) correspond to the S-, early G2-, late G2-, and M-phases andtogether contain 65% of all the tags. An additional cluster (cluster 5in FIG. 1C), not clearly separated in the hierarchical clustering,includes the genes with peak expression in G1-phase and contains another5% of the tags. The remaining clusters are much smaller and most often(e.g., clusters 6, 9, 10, and 18) include genes with a narrow temporalexpression pattern. In addition to these clusters, three small groups ofgenes displaying elevated expression during two cell cycle phases weredistinguished also by quality-based clustering (FIG. 1D).

After the transcript tags had been sequenced, homology searches revealedthat 36.5% of the tags were significantly homologous to genes of knownfunctions, 13.1% of the tags matched a cDNA or genomic sequence withoutallocated function, whereas for 50.4% of the tags no homology with aknown sequence was found. Genes of known function belong to diversefunctional classes (Table 1) revealing that several biological processesare at least partially under temporal transcriptional control during thecell cycle in plants. In general, the observed transcript accumulationprofiles and cell cycle specificity correlate well with the functionalproperties of the corresponding genes. It is interesting that the numberof transcription factors with G2-phase specificity is high, which may berelated with the induction of genes involved in M-phase-specificprocesses. The overrepresentation of RNA-processing genes in the M-phasemight indicate that post-transcriptional regulation is involved in geneactivity during mitosis. Because de novo transcription is severelyreduced during mitosis (Gottesfeld et al., Trends Bioch. Sci., 22197-202, 1997). RNA-processing could provide an alternative regulatorymechanism. Intriguingly, transcript tags with homology to a gene ofunknown function are overrepresented in the M-phase as well (Table 1).The principal differences in cell cycle events between plants and otherorganisms occur during mitosis; therefore, the inventors believe thatseveral of these transcripts correspond to still uncharacterisedplant-specific genes triggering these events. Remarkably, several of thetags homologous to a publicly available sequence have no Arabidopsishomologue, indicating that, in addition to conserved genes, differentplant species possess also unique sets of cell cycle-modulated genes.Although many of these tags may be too short to significantly match withan Arabidopsis sequence, analysis of longer cDNA clones corresponding toa subset of tags has revealed that approximately 25% of the sequencesremain novel.

In Tables 1 to 4 a selection of 785 sequence tags are shown. Thisselection was based on the criterion if the tags were full length orthat showed homology with genes known to be involved in the cell cycle(group 2 SEQ ID NOs 22 to 118), or on the criterion that they showhomology with genes of unknown function (group 3 SEQ ID NOs 119 to 283)or on the criterion that the sequences showed no homology with thesequences in that existing databases (group 4 SEQ ID NOs 284-785). Afirst group (SEQ ID Nos 1 to 21) represent a smaller selection of tagswhich are used in the target validation method described in the presentinvention, more particularly, that were used in example 2.

The Core Cell Cycle Machinery

Several tags coincide with genes belonging to the core cell cyclemachinery and exhibiting distinct expression profiles. Transcript tagsfrom five B1- or B2-type cyclins as well as from a D2-type cyclin showmitotic accumulation and exhibit a narrow temporal expression profile,confirming previous studies (Mironov et al., Plant Cell, 11 509-521,1999; Sorrell et al., Plant Physiol., 119 343-351, 1999). Based on thetranscription patterns, the six A-type cyclins fall into three groupsthat sequentially appear during the cell cycle, adding new data toearlier observations (Reichheld et al., PNAS, 93 13819-13824, 1996). Twogroups have quite a broad window of transcript accumulation; one group,homologous to A3-type cyclins, is expressed during S-phase anddisappears during G2-phase and the other group, corresponding to A2-typecyclins comes up at mid S-phase and goes down during M-phase, except forone transcript that is specific for S-phase. The third group, containingan A1-type cyclin, has the same expression pattern as the B- and D2-typecyclins. Several tags derived from genes encoding the plant-specificB-type cyclin-dependent kinases (CDKs) were also identified. CDKB1 andCDKB2 peak at the G2-to-M transition, slightly before the mitoticcyclins as describe (Porceddu et al., J. Biol. Chem., 276 36354-36360,2001). In contrast to what has been observed in partially synchronizedalfalfa cell cultures (Magyar et al., Plant Cell, 9 223-235, 1997), thetranscript levels of the tags homologous to a C-type CDK accumulatedifferentially during the cell cycle. The transcripts are present duringlate M-phase and early S-phase, suggesting that CDKC is active duringthe G1-phase.

In addition to these well-characterized cell cycle-regulatory genes,also several tags were identified herein derived from genes encodingtranscription factors and protein kinases or phosphatases with a knownor putative role in cell cycle control. One tag with a sharp peak oftranscript accumulation 1 h before the B- and D-type cyclins correspondsto a 3R-MYB transcription factor. Recently, a 3R-MYB has been shown toactivate B-type cyclins and other genes with a so-calledM-phase-specific activator domain (Ito et al., Plant Cell, 13 1891-1905,2001). Another tag peaking in M-phase is homologous to the CCR4associated protein CAF. CAF forms a complex with CCR4 and DBF2,resulting in a transcriptional activator involved in the regulation ofdiverse processes including cell wall integrity, methionine biosynthesisand M-to-G1 transition (Liu et al., EMBO J., 16 5289-5298, 1997). Amajority of the tags with similarity to protein kinases and phosphatasesshow M-phase-specific accumulation (Table 1). Although the true identityand putative cell cycle related function remains unclear for themajority, one is highly homologous to a dual-specificity phosphatase.This type of phosphatases plays a crucial role in cell cycle control inyeast and animals (Coleman and Dunphy, Curr. Opin. Cell Biol., 6877-882, 1994). Another M-phase-specific tag is homologous toprohibitin. In the mammalian cell cycle, prohibitin repressesE2F-mediated transcription via interaction with retinoblastoma (Rb),thereby blocking cellular proliferation (Wang et al., Oncogene, 183501-3510, 1999).

Protein degradation by the ubiquitin-proteasome pathway also plays animportant role in the control of cell cycle progression at both G1-to-Stransition and exit from mitosis. Although there is little evidence forcell cycle-modulated expression of the genes encoding the variouscomponents of the ubiquitin-proteasome complexes, some proteinsaccumulate in a cell cycle-dependent way (del Pozo and Estelle, PlantMol. Biol., 44 123-128, 2000). Furthermore, several tags were isolatedherein from genes encoding ubiquitin-conjugating enzyme (E3),ubiquitin-protein ligase (E2), and proteasome components with anM-phase-specific expression pattern. Another transcript tag thataccumulates during late M-phase is similar to cathepsin B-like proteins,which are proteolytically active and degrade diverse nuclear proteins,including Rb (Fu et al., FEBS Lett., 421 89-93, 1998).

Whereas all the core cell cycle regulatory genes have been identifiedthat control the G2-to-M transition for which the expression is known tobe cell cycle modulated, genes such as Rb and E2F, controlling G1-to-Stransition were not found. These genes were probably missed because theG1-to-S transition was not included in the present analysis, what issupported by the finding that the early targets of E2F, such aspolymerase α and ribonucleotide reductase, are already present at highlevels at the beginning of the time course.

Genes Involved in DNA Replication and Modification

In agreement with the studies performed in yeast and human fibroblasts,transcripts encoding proteins involved in DNA replication andmodification accumulated during S-phase and exhibited broad temporalexpression profiles. Different replication factors, DNA polymerase (α,and the histones H3 and H4 are already present at the onset of the timecourse, indicating that they are induced before the time point of theaphidicolin arrest. Interestingly, most of the histones H1, H2A, and H2Bappear somewhat later than H3 and H4, what might reflect that they aredeposited into the nucleosomes after H3 and H4 (Luger et al., Nature,389 251-260, 1997; Tyler et al., Nature, 402 555-560, 1999). The profileof the homologue of the anti-silencing function 1 (ASF1) protein issimilar to that of the histones H3 and H4, in agreement with the factthat the three proteins are part of the replication-coupling assemblyfactor complex that mediates chromatin assembly (Tyler et al., Nature,402 555-560, 1999). Genes encoding high-mobility group proteins reachthe highest accumulation during late G2, consistent with the subsequentsteps involved in the folding and structuring of the chromatin. Tagsderived from genes encoding proteins involved in DNA modification, suchas S-adenosyl-L-methionine (SAM) synthase andcytosine-5-methyl-transferase are found in the histone cluster. Tagsfrom methionine synthase genes, which provide the precursor for SAMsynthase, accumulate during M-phase, in contrast to yeast, where thesegenes are expressed during late S-phase (Spellman et al., Mol. CellBiol., 9 3273-3297, 1998).

Genes involved in chromatin remodelling and transcriptional activationor repression have been identified as well. One gene is a histonedeacetylase with highest transcript accumulation during the G2-phase andanother belongs to the SNF2 family of chromodomain proteins with anM-phase-specific expression pattern. Interestingly, one tag correspondsto a mammalian inhibitor of growth 1 (p33-ING1) protein. The human ING1protein has DNA-binding activity and might be involved inchromatin-mediated transcriptional regulation (Cheung and Li, Exp. CellRes., 268 1-6, 2001). This protein accumulates during S-phase(Garkavtsev and Riabowel, Mol. Cell Biol., 17 2014-2019, 1997), what isin agreement with the expression profile we observed. The yeasthomologues of ING1 are components of the histone acetyltransferasecomplex and show similarity to the Rb-binding protein 2 (Loewith et al.,Mol. Cell Biol., 20 3807-3816, 2000). Another tag, homologous to theArabidopsis MS13 protein, follows a similar expression profile. MSI-likeproteins are involved in the regulation of histone acetylation anddeacetylation and in chromatin formation (Ach et al., Plant Cell, 91595-1606, 1997).

The expression profiles of the different ribonucleotide reductase (RNR)genes are more complex. One gene is already expressed at high levels atthe beginning of the time course and its expression is restricted to theS-phase as described (Chaboute et al., Plant Mol. Biol., 38 797-806,1998), whereas, in contrast, another one is highly expressed in S-phaseand reappears at lower levels during M-phase and a third one isM-phase-specific. This latter expression profile has also been describedfor a RNR gene from Xenopus where the encoded protein appears to beinvolved in microtubulin nucleation (Takada et al., Mol. Cell Biol., 114173-4187, 2000).

Numerous other transcript tags with S-phase specificity were found inaddition to the ones involved in DNA replication and modification. Mostinterestingly, one of these tags is homologous to a mammalian geneencoding a TRAF-interacting protein (TRIP), which is a component of thetumor necrosis factor (TNF) signalling complex, and promotes cell deathwhen complexed with TRAF (Lee et al., J. Exp. Medicine, 185 1275-1285,1997). Another S-phase-specific tag shows homology to the RING fingerdomain of inhibitor of apoptosis proteins, which are also involved inthe TNF signalling pathway.

Modulated Expression of Genes Required for Mitosis and Cytokinesis

Several paralogous genes that encode either α- or β-tubulin were highlyinduced and accumulated prior to the mitotic index peak or during earlyM-phase. The inventors found that in BY2, tubulin genes are highly cellcycle modulated. This transcriptional regulation is in agreement withprevious demonstrations of de novo transcription of α- and β-tubulingenes during different cellular processes (Stotz et al., Plant Mol.Biol., 41 601-614, 1999). In the present analysis, no γ-tubulin geneswere found, confirming published data that the amount of γ-tubulin isconstant in dividing BY2 cells (Stoppin-Mellet et al., Plant Biol., 2290-296, 2000). Most of the kinesins identified herein, fall in the samecluster as the tubulins peaking prior to mitosis. Interestingly, twotags have a distinct transcription pattern and appear in another genecluster. Their window of transcript accumulation is very narrow andcoincides with the peak of mitosis. Most interestingly, these tagscorrespond to the plant-specific phragmoplast-associated type ofkinesin, PAKRP1 (Lee and Liu, Curr. Biol., 10 797-800, 2000). Achromokinesin not yet described in plants was identified as well. Thistype of motor proteins use DNA as cargo and play a role in chromosomesegregation and metaphase alignment (Wang et al., J. Cell Biol., 128761-768, 1995).

Among the M-phase-specific kinases, two were unambiguously recognizedherein as playing a role in cytokinesis. One is Aurora, a protein kinasewith a key role in the control of chromosome segregation, centrosomeseparation, and cytokinesis in yeast and animals (Bischoff and Plowman,Trends Cell Biol., 9 454-459, 1999) but not described in plants yet. Theother is NRK1, a mitogen-activated protein kinase kinase which isphosphorylated by NPK1, a kinase involved in regulating the outwardredistribution of phragmoplast microtubules (Nishihama et al., GenesDev., 15 352-363, 2001).

Hormonal Regulation and Cell Cycle-Modulated Gene Expression

A number of genes belonging to the class of auxin-induced genes werealso differentially expressed. Cell cycle-modulated expression ofauxin-induced genes has never been observed before although auxinstogether with cytokinins are the two major groups of plant hormones thataffect cell division (Stals and Inzé, Trends Plant Sci., 6 359-364,2001). The genes as identified herein fall into two groups based ontheir transcript accumulation profiles (data not shown). The first groupdisplays an early S-phase-specific expression pattern and consists ofthe parA, parB and parC genes. Induction of the par genes is most oftenobserved in response to stress conditions (Abel & Theologis, Plant Phys.111, 9-17, 1996). The fact that the transcripts rapidly disappear afterrelease from the cell cycle-blocking agent might indicate a stressresponse rather than a cell cycle dependent auxin response.

More interesting is the second group of genes with transcriptsaccumulating during early M-phase. This group includes the auxinresponse factor 1 (ARF1), an auxin transporter as well as differentmembers of the early auxin response AUX/IAA gene family. ARF1 is atranscription factor that binds to a particular auxin response element(Ulmasov et al., Science, 276 1865-1868, 1997). Additional studiessuggest that the activity of ARF1 is controlled by its dimerization withmembers of the AUX1/IAA family (Walker and Estelle, Curr. Opin. Plantboil., 1 434-439, 1998). The similarity in temporal expression profilesthe inventors observed supports these findings and suggests that theseproteins mediate an auxin response necessary for cell cycle progression

By using tobacco BY2 as model system together with cDNA-AFLP-basedtranscript profiling, it is described herein for the first time how acomprehensive inventory of plant cell cycle-modulated genes can be made.Although the obtained data confirm earlier results and observations, inaddition, numerous novel findings were made. The obtained data are avery useful basis for selecting and validating agrochemical targetgenes.

Example 2

In this example it is described how plant genes are evaluated forassessment of their essential character in the biological process, thushow they are validated as good candidate targets for agrochemicals.

The Tobacco Rattle Virus (TVR) is used to induce silencing of targetgenes. In case of an essential gene the simlencing will result in alethal effect on the plant and therefore, the suystem allows to validategood candidates as targets for herbicides.

The TRV based system is used in this example in combination with seriesof candidate genes, more particularly with the candidate targets asrepresented herein as group 1 sequences consisting of the SEQ ID NOs 1to 21. The identification technique of the present invention (seeexample 1) allowed to identify new genes that are potential newherbicide targets, because of their putative function in various keyprocesses crucial for cell life, their expression at a certaindevelopmental stage crucial for cell life, their role in metabolismand/or maintenance of cell living state.

This example illustrates the validation of these candidate genes asnovel targets for agrochemicals, via the technique of the virus-inducedgene silencing (VIGS).

Gene Silencing Mechanism

The virus-induced gene silencing (VIGS) is a manifestation of anRNA-mediated defence mechanism that is related to post-transcriptionalgene silencing (PTGS) in transgenic plants (Ratcliff et al., Plant J.,25 237-245, 2001). The method uses a vector with an infectious cDNA oftobacco rattle virus (TRV) modified (see below) to facilitate insertionof target sequences and modified for efficient infection of plants (e.g.tobacco). The vector mediates VIGS of endogenous genes in the absence ofspecific virus-induced symptoms.

The RNA-mediated defence is triggered by the virus vectors, and targetsboth the viral genome and the host gene corresponding to the insert. Asa result, the symptoms in the infected plant are similar toloss-of-function mutants or reduced-expression mutants in the host gene.The presence of a negative growth phenotype suggests that the targetedgene is a potential herbicide target.

The process of constructing a virus vector and monitoring symptoms oninfected plants is completed within a few weeks, such that virus-inducedgene silencing (VIGS) provides a simple, rapid means of assigningfunction to genes that have been sequenced but are otherwiseuncharacterized. The determination of new herbicide target genes isperformed in a few weeks including gene cloning, transformation stepsand tobacco plant analyses.

The TRV construct is shown to target host RNAs in the growing points ofplants (Ratcliff et al., Plant J., 25 237-245, 2001) such as meristemsand actively dividing cells. It has been shown that this vectorovercomes many of the problem features of PVX, TMV and TGMV. Forexample, the TRV vector induces very mild symptoms, infects large areasof adjacent cells and silences gene expression in growing points such asmeristems and actively dividing cells. Infection of tobacco plants onthe leaves with TRV based constructs will affect growth and developmentof upper parts of the infected leaves and allow screening for growthparameters.

Construction of TRV Vectors Used in the Validation Process of thePresent Invention

TRV is a positive-strand RNA virus with a bipartite genome. Proteinsencoded by RNA 1 are sufficient for replication and movement within thehost plant, while proteins encoded by RNA 2 allow virion formation andnematode-mediated transmission between plants (reviewed by MacFarlane,J. Gen. Virol., 80 2799-2807,1999).

The downregulation system is composed of separate cDNA clones of TRV RNA1 and RNA 2 under the control of cauliflower mosaic virus (CaMV) 35Spromoters on the transferred T-DNA of plant binary transformationvectors.

The TRV RNA 1 construct (pBINTRA6) contains a full-length infectiouscDNA clone in which the RNA polymerase ORF is interrupted by intron 3 ofthe Arabidopsis Col-0 nitrate reductase NIA1 gene (Wilkinson andCrawford, Mol. Gen. Genet., 239 289-297, 1993), necessary to preventexpression of a TRV-encoded protein that is toxic to E. coli. Thisvector has been given the internal reference number p3209.

The TRV RNA 2 construct (pTV00), contains a multiple cloning site (MCS),leaving only the 5′ and 3′ untranslated regions and the viral coatprotein (Ratcliff et al., Plant Cell, 11 1207-1215, 1999). This vectorhas the internal reference number p3930 and contains a Gateway™ cassetteand the gene of interest to be tested. The genes as presented in SEQ IDNO 1 to 21 are each cloned in this vector.

cDNAs were amplified using Gateway compatible primers and the cDNAs wereentered into Entry Clones by BP recombination reactions. Subsequentlythe entry clones comprising the gene according to any one of SEQ ID NO 1to 21 were checked via Ban2 restriction digest. The genes of interestwere then entered into destination vectors by LR recombination reactionsand the destination vectors were checked via ECORV restrictiondigestions. These expression clones were electroporated into theArgobacterium strain GV3101 agro and the plasmid pBintra6 waselectroporated into pMP90 agro.

Inoculation

To inoculate plants, Agrobacterium cultures carrying pBINTRA6 (strainC58C1RifR containing pMP90 plasmid) and pTV00 (strain GV3101 containingpMP90 plasmid) were grown and mixed and infiltrated to the leaves ofNicotiana benthamiana as previously described (English et al., Plant J.,12 597-603, 1997). Briefly, virus infection was achieved byAgrobacterium-mediated transient gene expression. Agrobacteriumcontaining the TRV cloning vectors were grown overnight in L brith(+Tc+Km), Agrobacterium containing the helper plasmid was grownovernight in 10 ml YEB+Rif+Km. The culture was centrifuged andresuspended in 10 ml of 10 mM MgCl₂, 1 mM MES-pH5.6 and 100 μMacetosyringone and kept at room temperature for 2 h. Separate culturescontaining pBINTRA6 and TRV cloning vectors were mixed in a ratio of1:10. The culture was then infiltrated to the underside of two leaves ofthree-weeks old plants using a 2 ml syringe without a needle. In twoindependent experiments 6 plants per agroabcterium clone were infected.In this way the cloned genes (SEQ ID NO 1-21) were transferred into thecells of the infiltrated region, and could be transcribed into the viralcDNAs in the leave cells. These transcripts then serve as an inoculum toinitiate systemic infection of the plant. Consequently the VIGS systemis activated, resulting in the downregulation of the host cell gene,corresponding to the cloned gene of interest. All experiments involvingvirus-infected material was carried out in controlled growth chambers.N. benthamiana plants were germinated ad grown individually on universalpotting ground in pots at 25° C. during the day (16 h) and 20° C. duringthe night (8 h).

The plants were phenotypically evaluated on a daily basis. Particularattention was given to visible leaf damage and growth inhibition. Theeffects of the suppression of gene activity using the VIGS system ismeasured by the phenotypic aspect of the plants, including leaf defectssuch as growth retardation, yellow or necrotic spots, early senescence,etc. The effects of the downregulation of genes identified by themethods of the invention are also measured on the flower structure andthe flowering capacities of the transformed plants.

The severity of the phenotype is linked to the level of suppression ofthe gene activity and indicates the degree in which the gene isessential for the plant Therefor the phenotype is an indication of thedegree in which the gene is a valid target for a herbicide.

Phenotypes of the Infected Plants.

1. Co-suppression of the gene leads to loss of gene transcription andprotein expression in the virus infected leaf and induces leaf growthmodification, including leaf wrinkling, curling, wilting, leading tocell death and/or plant death.

2. Co-suppression of the geneleads to loss of gene transcription andprotein expression in the virus infected leaf and induces leaf yellowingor senescence, or cell death and necrosis, leading to plant death.

3. Co-suppression of the gene leads to loss of gene transcription andprotein expression in the virus infected leaf and induces any of thefollowing phenotypic symptoms: chlorotic regions around infection, crispor crunchy leaf texture around infection, numerous surface lumps oneither leaf surface, abnormal trichomes, abnormal leaf size, reducedgrowth, reduced final size, altered vascular leaf system, altered watermovement in leaf, leading to cell death and/or plant death.

4. Co-suppression of the gene leads to loss of gene transcription andprotein expression in the virus infected leaf and induces any of thefollowing anatomical symptoms: clumps of modified cells on the surfaceof the leaf (either abaxial or adaxial), individual cells detached fromthe epidermis, swollen or modified trichome cells, modification of leaftissue structure, cell size, cell number, tissue composition,parenchyme, epidermis, etc , leading to cell death and/or plant death.

5. co-suppression of gene X leads to loss of gene transcription andprotein expression in the virus infected leaf and induces any of thefollowing biochemical symptoms, enzyme activity and products,degradation of leaf components and effects in neighboring leaves, stem,vascular system, degradation of cell wall structure, communicationbetween cells, modification of cell-cell signaling leading to cell deathand/or plant death.

The genes identified by the present invention can be utilized to examineherbicide tolerance mechanisms in a variety of plants cells, includinggymnosperms, monocots and dicots. It is particularly useful in cropplant cells such as rice, corn, wheat, barley, rye, sugar beet, etc

Example 3

Significant phenotypic alterations could be observed in plantsinfiltrated with Agrobacterium containing pBINTRA6+Bstt44-4-340 (SEQ IDNO 18, acetolactate synthetase) and pBINTRA6+Bstt2-42-520 (or T4-32-7)(SEQ ID NO 21, prohibitin) and pBINTRA6+Bstt23-4-230 (SEQ ID NO 11,B-type CDK).

At 10 days post-infiltration the first symptoms were visible. Thesymptoms were persistent until the end of the experiment and could beobserved in at least 5 out of the 6 infiltrated plants.

The phenotypes of the plants transformed with acetolactate synthase arefurther described. In two separate replicated experiments, specificphenotypes on each plant infected with the acetolactate synthetasedownregulation construct were observed (FIG. 2). Winkling and wrappingof the leaves as well as some chlorotic spots were observed. Thusacetolactate downregulation provoked a general growth arrest accompaniedwith chlorotic and necrotic areas. These observations were in line withprevious reports, wherein acetolactate synthetase is described as auseful herbicide target.

The phenotypes of the plants transformed with prohibitin are furtherdescribed.

In two separate replicated experiments, specific phenotypes on eachplant infected with the prohibitin downregulation construct wereobserved (FIG. 2). These plants showed strong wrinkling of the leavesabout 20 days after infection, corresponding to the expected occurrenceof silencing events. Thus the downregulation of probibitin provokes asevere leaf distortion and general growth arrest.

The phenotype of the plants inoculated with a B-type CDK downregulationconstruct are shown in FIG. 3. A late (from 30 days after inoculation)but strong negative effect on the plant growth was observed. The plantsstarted to grow much slower and lost their apical dominance, resultingin the increased appearance of lateral branches. TABLE 1 Functionalclassification of transcript tags S G2 M G1 Function Tags 27.7% 15.8%52.9% 3.6% Cell cycle control 30 5/8 (0.078) 8/5 (0.068) 14/16 (0.114)3/1 Cell wall 35 6/10 (0.047) 4/6 (0.136) 25/18

0/1 Cytoskeleton 43 1/12 (1.2e⁻⁵) 4/7 (0.090) 38/22

0/2 Hormone response 13 6/4 (0.113) 1/2 (0.277) 6/7 (0.185) 0/0Kinases/phosphatases¹ 27 4/8 (0.039) 1/4 (0.059) 19/14 (0.025) 3/1Protein synthesis 50 15/14 (0.116) 5/8 (0.087) 29/26 (0.079) 1/2Proteolysis 21 2/6 (0.026) 1/3 (0.144) 17/11 (0.039) 1/1 Replication andmodification 74 57/20

8/12 (1.0e⁻⁵) 8/39 (1.0e⁻¹⁸) 1/3 RNA processing 20 1/6 (6.8e⁻³) 1/3(0.137) 18/11

0/0 Signal transduction 10 1/3 (0.121) 3/2 (0.201) 6/5 (0.205) 0/0Stress response 20 6/6 (0.192) 2/3 (0.229) 10/10 (0.159) 2/1Transcription factors 27 4/8 (0.039) 10/4

12/14 (0.112) 1/1 Transport and secretion² 31 5/9 (0.047) 2/5 (0.076)21/16 (0.031) 3/1 Unknown 175 37/48 (0.015) 19/28 (0.014) 112/93

7/6¹Only kinases and phosphatases with unknown biological function.²Except small GTP-binding proteins, which are classified under signaltransduction.

The total number of tags and the observed/expected number of tags withinthe different cell cycle phases for each functional group is giventogether with the probability values between parentheses as calculatedbased on the binomial distribution function, except for the G1-phasebecause the values were too small. A significant enrichment (P<e⁻³) oftags of a functional group within a particular cell cycle phase isindicated in bold. TABLE 2 overview of group 1 of sequences used forvalidation of candidate target genes SEQ ID NO CDS NO Tag Name FunctionFase 1 2216 18R1850_C4-32-33_1E2 catalase ?? 2 2217 Bstt2-31-215phytoene desasturase ?? 3 2218 Bstc13-1-145 L-ascorbate peroxidase M-G14 2219 Bstc21-4-280 GTP-bindingprotein M 5 2220 Bstc33-2-310vacuolarsortingreceptor M 6 2221 Bstc4-34-170 probable cinnamyl alcoholdehydrogenase G1/S—S; M-G1 7 2222 Bstt34-3-470 kinesin M 8 2223Bstt12-3-410 B-typeCDK M 9 2224 Bstt14-3-458 squalene mono-oxygenaseG1/S—S 10 2225 Bstt12-1-230 kinesin-likeprotein M 11 2226 Bstt23-4-230B-typeCDK M 12 2227 Bstt2-42-225 B-typeCDK M 13 2228 Bstt31-4-208arabinogalactan protein precursor G2/M—M 14 2229 Bstt3-41-205arabinogalactan protein precursor G2/M—M 15 2230 Bstt33-4-285 chorismatesynthase S-G2 16 2231 Bstt2-31-215 kinesin-likeprotein M 17 2232Bstt41-2-400 endo-beta-1,4glucanase M 18 2233 Bstt44-4-340 acetolactatesynthase G2/S-G2-M-G1 19 2234 G17-2-13 G17-2-13 WRKY transcriptionfactor ?? 20 2235 mapk9-ntf6.seq mapkinase phragmoplast associated NTF6?? 21 2236 Bstt2-42-520 prohibitin ??

TABLE 3 overview of group 2 sequences of full-length sequences that arecell cycle modulated and of which some are involved in the cell cycleprocess SEQ ID CDS NO NO Gene name 22 0613 Protein kinase mRNA,complete, N. tabacum, 2073 bp 23 0614 BY2 AA041K03 probable DNA-bindingprotein GBP16 - rice T02069, N. tabacum, 834 bp 24 0615 BY2 AA042C09probable nuclear DNA-binding protein G2p [imported] in ArabidopsisT51151, N. tabacum, 1185 bp 25 0616 BY2-AA044J17 transcriptionregulator-like in Arabidopsis AB025604, N. tabacum, 1893 bp 26 0617 BY2AA044J23 ATP-dependent RNA helicase CA3 of the DEAD/DEAH box family;Dbp3p; BY2- AA044J23P19G01 RNA helicase RH5 in Arabidopsis T51739 N.tabacum, 1593 bp 27 0618 BY2-AA046C15 protein phosphatase 2C-like inArabidopsis BAB08417 AB025622, N. tabacum, 732 bp 28 0619 BY2-AA047G1314-3—3-like protein C P93343, N. tabacum, 70 bp 29 0620 BY2-AA054L09protein kinase tousled in Arabidopsis A49318 N. tabacum, 2037 bp 30 0621BY2-AA066H11P19H05 phosphoprotein phosphatase 2A regulatory chain T03684N. tabacum, 1764 bp 31 0622 BY2-AA069L10 transcription factor-likeprotein in Arabidopsis BAB09482 AB012246, N. tabacum, 831 bp 32 0623BY2-AA073K06 SET protein, phospatase 2A inhibitor in ArabidopsisAAG52377.1 AC011765, N. tabacum 33 0624 BY2-AA073MP19B07 phosphoproteinphosphatase 2A regulatory chain T03684, N. tabacum, 1764 bp 34 0625BY2-AA075H12 Putative phospatase 2A inhibitor in Arabidopsis AC011809_9AC011809, N. tabacum, 783 bp 35 0626 BY2-AA076O02P19B08 hypotheticalprotein kinase in Arabidopsis T47727, N. tabacum, 2514 bp 36 0627BY2-AA079J13 putative casein kinase | in Arabidopsis AAG51841.1AC010926_4, N. tabacum, 1401 bp 37 0628 BY2-AA080G14 porin I 36K inpotato S46959, N. tabacum, 393 bp 38 0629 BY2-AA081P13p21E02 separationanxiety protein-like in Arabidopsis CAB96669.1 AL360314, N. tabacum, 492bp 39 0630 Complementary copy of 0630, N. tabacum, 975 bp 40 0631BY2-AA085N17p21H04 14-3—3-like protein in potato 16R P93784 N. tabacum768 bp 41 0632 BY2-AA087C16p21G03 AP2 domain transcription factorhomolog in potato T07784 N. tabacum, 891 bp 42 0633 BY2-AA088B13putative RING zinc finger protein in Arabidopsis CAB80936.1 AL161491 N.tabacum 1248 bp 43 0634 BY2-AA095M08 protein kinase homolog inArabidopsis T02181 N. tabacum858 44 0635 BY2-AA096M07 peptidyl-prolylcis-trans isomerase-like protein BAB10691.1 AB015468 N. tabacum 450 bp45 0636 BY2-AA096M12 zinc finger protein-like in Arabidopsis BAB09106.1AB017069 N. tabacum 1518 bp 46 0637 BY2-AA096M22 cell division-likeprotein in Arabidopsis T45963 N. tabacum 687 bp 47 0638_1BY2-AA098B08p21D11 similarity to DAG protein in Arabidopsis BAA97063.1AP000370 N. tabacum 1146 bp 48 0638_2 Icl_AA091G16p21F05 N. tabacum 891bp 49 0639 BY2-AA109N15 GAMM1 protein-like in Arabidopsis BAB08430.1AB017067 N. tabacum 888 bp, (MYG1) FAMILY, proliferation associated 500640 Complementary copy of 0640 N. tabacum, 891 bp 51 0641 BY2-AA114N16unknown protein in Arabidopsis BAB03019.1 AP001297; candidate tumorsuppressor p33 ING1 homolog in Homo sapiens N. tabacum 720 bp 52 0642BY2-AA115P21p22D02 NAC2 Arabidopsis AAF09254.1 AF201456_1N. tabacum 699bp 53 0643 BY2-AA119N11p22G04 serine/threonine-specific proteinkinase-like protein BAB09338.1 AB016879 N. tabacum 1293 bp 54 0662BY2-AA041E04 >pir∥T06678 hypothetical protein T17F15.80 - Arabidopsisthaliana 55 0663 BY2-AA043A01 >gb|AAD24540.1|AF113545_1 (AF113545)vacuole-associated annexin VCaB42 [Nicotiana tabacum] 56 0664BY2-AA044C02 >dbj|BAA02028.1| (D11470) chloroplast elongation factorTuB(EF-TuB).[Nicotiana tabacum] 57 0665 BY2-AA044L14 dbj|BAA97319.1|(AB020754) gene_id:MYN8.3˜pir∥T02891˜similar to unknown protein 58 0666BY2-AA045P04p01G10 sp|Q43681|NLTP_VIGUN PROBABLE NONSPECIFICLIPID-TRANSFER PROTEIN AKCS9 59 0667 BY2-AA046C08p19E02 dbj|BAB30364.1|(AK016659) putative [Mus musculus] 60 0668 BY2-AA046E06 pir∥T50556stamina pistilloidia protein Stp [imported] - garden pea 61 0669BY2-AA046G14 dbj|BAB26082.1| (AK009117) putative [Mus musculus] 62 0670BY2-AA046H23 emb|CAA98172.1| (Z73944) RAB8A [Lotus japonicus] 63 0671BY2AA048A05 gb|AAD15504.1| (AC006439) putativeAAA-type ATPase[Arabidopsis thaliana] 64 0672 BY2-AA049K03 dbj|BAB24909.1| (AK007240)putative [Mus musculus] 65 0673 BY2-AA051A10 dbj|BAB02543.1| (AP000417)mitotic checkpoint protein [Arabidopsis thaliana] 66 0674BY2-AA051L22p19H03 gb|AAD48948.1|AF147262_11 (AF147262) containssimilarity to Pfam family PF00400-WD domain 67 0675BY2-AA052E10 >gb|AAF52905.1| (AE003628) CG4968 gene product [Drosophilamelanogaster] 68 0676 BY2-AA052F14 >gb|AAF79819.1|AC007396_20(AC0007396) T4O12.22 [Arabidopsis thaliana] 69 0677BY2-AA052G16p19D04 >dbj|BAB09843.1| (AB005246) gene_id: MUP24.12˜unknownprotein [Arabidopsis thaliana] 70 0678BY2-AA052N17 >gb|AAG42914.1|AF327533_1 (AF327533) unknown protein[Arabidopsis thaliana] 71 0679_1 BY2-AA053C11.1 >dbj|BAB22857.1|(AK003561) putative [Mus musculus] 72 0679_2BY2-AA053C11.2 >gb|AAC62883.1| (AC005397) hypothetical protein[Arabidopsis thaliana] 73 0680 BY2-AA062A09 >gb|AAF01061.1|AF189284_1(AF189284) nucleolar G-protein NOG1 [Trypanosoma brucei] 74 0681BY2-AA062G03 >pir∥T02135 hypothetical protein F8K4.10 - Arabidopsisthaliana 75 0682 BY2-AA065E08 >pir∥T00795 hypothetical proteinF24L7.13 - Arabidopsis thaliana 76 0683 BY2-AA072K18 >emb|CAB40381.1|(AJ010819) GrpE protein [Arabidopsis thaliana] 77 0684BY2-AA075K12 >gb|AAD31331.1|AC007354_4 (AC007354) T16B5.4 [Arabidopsisthaliana] 78 0685 BY2-AA076N08 >dbj|BAA94770.1| (AP001859) ESTsAU082761(S5084) D42006 79 0686 BY2-AA080D01 >gb|AAF80646.1|AC012190_2(AC012190) Contains similarity to F28O16.19 a putative translationinitiation protein 80 0687 BY2-AA081P14 >gb|AAD32777.1|AC007661_14(AC007661) unknown protein [Arabidopsis thaliana 81 0688BY2-AA082H04p21F02 >dbj|BAB10171.1| (AB016880) gene_id:MTG10.12˜pir∥T05795˜strong similarity to unknown 82 0689BY2-AA082H06p21G04 >pir∥T09039 hypothetical protein F26K10.110 -Arabidopsis thaliana 83 0690 BY2-AA082M07p21B05 >dbj|BAB01783.1|(AB022215) gene_id: MCB17.19˜unknown protein [Arabidopsis thaliana] 840691 BY2-AA083B24p21C04 >dbj|BAB08247.1| (AB006698) gene_id:MCL19.6˜unknown protein [Arabidopsis thaliana] 85 0692BY2-AA083C05p21D02 >gb|AAH02924.1|AAH02924 (BC002924) Unknown (proteinfor IMAGE: 3956179) [Homo sapiens] 86 0693BY2-AA085D08p21C05 >pir∥T47624 hypothetical protein T5N23.10 -Arabidopsis thaliana 87 0694BY2-AA085F09p21H01 >gb|AAF79503.1|AC002328_11 (AC002328) F20N2.15[Arabidopsis thaliana] 88 0695BY2-AA085M15p21D04 >gb|AAF97305.1|AC007843_8 (AC007843) Unknown protein[Arabidopsis thaliana] 89 0696BY2-AA088K23p21G05 >gb|AAG52001.1|AC012563_11 (AC012563) unknownprotein; 64612-65506 [Arabidosis thaliana] 90 0697BY2-AA088L24p21A07 >gb|AAD55292.1|AC008263_23 (AC008263) ContainsPF|00249 Myb-like DNA- binding domain. 91 0698BY2-AA089F12p21H05 >gb|AAD55274.1|AC008263_5 (AC008263) Strongsimilarity to gb|D21805 calcium-dependent protein kinase 92 0699BY2-AA089M17 >pir∥T02186 hypothetical protein F14M4.16 - Arabidopsisthaliana 93 0700 BY2-AA090J23p21G08 >pir∥T48545 hypothetical proteinF14F18.30 - Arabidopsis thaliana 94 0701BY2-AA092F12p21H06 >emb|CAB46854.1| (AJ388555) hypothetical protein[Canis familiaris] 95 0702 BY2-AA092L20p21E07 >gb|AAD10646.1| (AC005223)45643 [Arabidopsis thaliana] 96 0703BY2-AA093J23p21C11 >gb|AAG51461.1|AC069160_7 (AC069160) unknown protein[Arabidopsis thaliana] 97 0704 BY2-AA093L18p21D09 >emb|CAC15504.1|(AJ297917) B2-type cyclin dependent kinase [Lycopersicon 98 0705BY2-AA093M19 >gb|AAG12535.1|AC015446_16 (AC015446) Unknown protein[Arabidopsis thaliana] 99 0706 BY2-AA094B12p21F10 >dbj|BAB02118.1|(AP000381) contains similarity to unknown 100 0707_1 BY2-AA096G05p21A11dbj|BAB02118.1| (AP000381) contains similarity to unknown 101 0707_2Icl_AA094B12p21F10 102 0708 BY2-AA097G22p21D10 >gb|AAG60065.1|AF337913_1(AF337913) unknown protein [Arabidopsis thaliana 103 0709 BY2-AA099F04gb|AAG52457.1|AC010852_14 (AC010852) hypothetical protein; 12785-11538[Arabidopsis thaliana] 104 0710 BY2-AA099N08p21H09gb|AAK14411.1|AC087851_3 (AC087851) unknown protein [Oryza sativa] 1050711 Icl_AA100B09 ref|NP_009820.1|Ybr261cp [Saccharomyces cerevisiae]106 0712 BY2-AA109N02 ref|NP_002848.1| peroxisomal farnesylated protein;Housekeeping gene 33 kD [Homo sapiens 107 0713 BY2-AA114E09p22F02pir∥T51434 hypothetical protein F2G14_10 - Arabidopsis thaliana 108 0714BY2-AA115B14p22C02 dbj|BAB08888.1| (AB012243) gene id:MIJ24.6˜ref|NP_013897.1˜similar to unknown protein 109 0715BY2-AA115F08p22C04 >gb|BY2-AAH03900.1|AAH03900 (BC003900) Similar tohypothetical protein 384D8_6 [Mus musculus] 110 0716BY2-AA115L12p22G01 >gb|AAF43925.1|AC012188_2 (AC012188) Containssimilarity to PIT1 from Arabidopsis thaliana 111 0717BY2-AA116L23p22E01 >dbj|BAB01460.1| (AP000731) gene_id: MCB17.21˜unknownprotein [Arabidopsis thaliana] 112 0718BY2-AA117B12p21G12 >sp|O23708|PSA2_ARATH PROTEASOME SUBUNIT ALPHA TYPE 2(20S PROTEASOME ALPHA SUBUNIT B) 113 0719 BY2-AA117E08p22A03 >pir∥F81195conserved hypothetical protein NMB0465 [imported] - Neisseria 114 0720BY2-AA117O08p22E03 >dbj|BAB01753.1| (AP000603)gb|BY2-AAD10646.1˜gene_id: MRP15.12 115 0721BY2-AA118D23p22E02 >emb|CAB89490.1| (AJ277062) CRK1 protein [Betavulgaris], cdc2 like kinase 116 0722 BY2-AA119D12p22H04 >dbj|BAB01163.1|(AP000410) gene_id: K10D20.9˜unknown protein [Arabidopsis thaliana] 1170723 BY2-AA120G12 >gb|BY2-AAB63649.1| (AC001645) hypothetical protein[Arabidopsis thaliana] 118 0724BY2-AA120G19p22D05 >gb|BY2-AAF69547.1|AC008007_22 (AC008007) F12M16.18[Arabidopsis thaliana]

TABLE 4 overview of group 3 sequences that show homology with proteinsof unknown function SEQ ID NO Tag name and Function Fase 119Bstc1-11-320 M-G1 120 Bstc1-12-255 G2/M-M-G1 121 Bstc1-12-275 G2/M-M-G1122 Bstc1-13-143 unknownprotein G2/M-M-G1 123 Bstc1-13-160unknownprotein G2/M-M-G1 124 Bstc11-3-190 M-G1 125 Bstc11-3-215putativeprotein G2/M-M-G1 126 Bstc11-3-230 G1/S; M-G1 127 Bstc11-3-300unknown M-G1 128 Bstc13-4-168 hypotheticalprotein S-G2 129 Bstc13-4-290hypotheticalprotein M-G1 130 Bstc14-205 G2/S-G2 131 Bstc1-43-107 G2/S-G2132 Bstc14-3-165 unknown M-G1 133 Bstc1-43-250 unknown G2/M-M-G1 134Bstc1-43-310 hypotheticalprotein G2/M-M 135 Bstc21-2-270hypotheticalprotein G2/M-M-G1 136 Bstc2-21-182 unknown M-G1 137Bstc22-1-275 unknownprotein G2-M-G1 138 Bstc2-22-100 unknown G2-G2/M 139Bstc2-22-155 G2-M 140 Bstc2-22-240 hypotheticalprotein M 141Bstc22-2-270 G1/S; M-G1 142 Bstc2-23-135 G2/S-G2-M 143 Bstc2-23-220unknown G2-M-G1 144 Bstc22-4-215 hypotheticalprotein G2/M-M 145Bstc2-31-280 G2/M-M-G1 146 Bstc23-2-240 unknown M 147 Bstc23-2-330putativeprotein M 148 Bstc23-2-370 G1/S-S; G2/M-M-G1 149 Bstc2-32-400G1/S-S; G2/M-M-G1 150 Bstc23-3-270 G1/S-S; -M-G1 151 Bstc2-33-280unknownprotein G1/S-S; M-G1 152 Bstc2-34-120 unknown G2/M-M-G1 153Bstc23-4-300 unknown M 154 Bstc2-41-165 G1/S-S 155 Bstc2-42-100 unknownG1/S-S 156 Bstc2-43-210 M-G1 157 Bstc31-185 unknown G2/M-M-G1 158Bstc3-12-145 unknown S-G2 159 Bstc3-12-290 unknown G2/M-M-G1 160Bstc31-3-400 unknown G2/M-M-G1 161 Bstc32-1-122 unknown M-G1 162Bstc3-21-125 G1/S-S; G2/M-M-G1 163 Bstc32-2-150 putativeprotein G1/S-S;G2/M-M-G1 164 Bstc32-4-193 165 Bstc32-4-370 G1/S-S-G2/S; M-G1 166Bstc3-31-350 putativeprotein G1/S-S-G2/S 167 Bstc33-2-145hypotheticalprotein G1/S-S; G2/M-M-G1 168 Bstc3-33-350 G1/S-S 169Bstc33-360 putativeprotein G2/M-M-G1 170 Bstc33-4-270 unknown G2/M-M 171Bstc3-41-270 unknown M-G1 172 Bstc3-41-300 G2/M-M-G1 173 Bstc3-41-360G2/M-M-G1 174 Bstc3-42-175 M-G1 175 Bstc3-43-135 G1 176 Bstc3-43-180M-G1 177 Bstc3-43-193 unknown G1/S-S; G2/M-M-G1 178 Bstc3-43-287 G1/SS179 Bstc3-44-145 M-G1 180 Bstc3-44-375 putativeprotein M-G1 181Bstc4-11-120 hypotheticalprotein G2/M-M-G1 182 Bstc4-11-320 unknown M-G1183 Bstc42-3-115 unknown M-G1 184 Bstc42-3-125 putativeprotein G2/M-M-G1185 Bstc4-23-210 M-G1 186 Bstc42-4-225 unknown G1/S-S-G2 187Bstc4-32-115 unknownprotein G1/S-S; G2/M-M-G1 188 Bstc4-32-185 unknownG1/S-S 189 Bstc4-32-190 unknown G2/M-M 190 Bstc4-32-270 unknownG2/S-G2-M 191 Bstc4-32-410 G1/S-S-G2- G2/M 192 Bstc4-34-250 G2/M-M-G1193 Bstc4-41-230 putativeprotein G2/M-M-G1 194 Bstc4-43-113 unknown M-G1195 Bstc44-3-125 G2/M-M 196 Bstt1-12-340 unknown G2/M-M 197 Bstt12-2-225G1/S-S-G2 198 Bstt1-22-330 unknown G2/M-M-G1 199 Bstt12-2-420unknownprotein G2/M-M-G1 200 Bstt12-2-540 hypotheticalprotein G2/M-M-G1201 Bstt1-23-155 M-G1 202 Bstt12-3-215 hypotheticalprotein G2/M-M-G1 203Bstt12-3-280 unknown G1/S-S-G2 204 Bstt12-3-310 hypotheticalproteinG1/S-S 205 Bstt12-3-350 G1/S-S-G2- G2/M 206 Bstt1-24-205 G2/M-M-G1 207Bstt1-24-220 G1/S-S-G2 208 Bstt1-31-170 hypotheticalprotein G2/M-M-G1209 Bstt1-31-215 unknown G2/M-M-G1 210 Bstt13-210 unknown G2/M-M-G1 211Bstt14-4-310 unknownprotein G2/M-M-G1 212 Bstt2-11-165 unknown G2/M-M-G1213 Bstt2-12-190 G1/S-S-G2 214 Bstt21-4-150 hypotheticalproteinG1/S-S-G2/S 215 Bstt21-4-250 G1/S-S; G2/M-G1 216 Bstt21-4-470 G2/M-M-G1217 Bstt22-1-170 unknown S-G2 218 Bstt2-21-190 unknown G2/M-M 219Bstt22-2-190 unknown G2/M-M-G1 220 Bstt22-2-290 hypotheticalproteinG2/M-M-G1 221 Bstt22-3-225 M 222 Bstt22-3-275 unknown G2/M-M 223Bstt22-3-315 TomatoEST G2/M-M-G1 224 Bstt22-3-370 unknown G2/M-M-G1 225Bstt22-3-390 putativeprotein G2/M-M-G1 226 Bstt22-3-480 G2/M-M-G1 227Bstt23-1-140 S-G2-G2/M 228 Bstt23-120 unknownprotein G2/M-M-G1 229Bstt23-1-200 S-G2-M 230 Bstt2-31-300 unknown S 231 Bstt2-32-220 M 232Bstt2-32-400 hypotheticalprotein G2/M-M-G1 233 Bstt23-3-350 unknown G2-M234 Bstt23-370 unknown G2/M-M-G1 235 Bstt24-1-320 S-G2 236 Bstt24-2-310G2/M-M-G1 237 Bstt2-43-210 unknown G2-M 238 Bstt2-43-240 S-G2/S 239Bstt31-1-100 hypotheticalprotein G1/S-S-G2 240 Bstt3-11-205 G1/S-S-G2241 Bstt31-1-250 hypotheticalprotein G2/M-M-G1 242 Bstt31-1-430hypotheticalprotein G2/M-M-G1 243 Bstt3-12-360 unknownprotein G2/M-M 244Bstt31-3-380 G1/S-S 245 Bstt31-4-420 hypotheticalprotein G2/M-M-G1 246Bstt32-180 putativeprotein G2-M-G1 247 Bstt3-22-160PotatoEST/hypothetical G1/S-S-G2 protein 248 Bstt32-3-175 unknown G2/M-M249 Bstt32-3-325 unknown protein G2/M-M-G1 250 Bstt3-24-135 unknownG2/M-M-G1 251 Bstt3-24-200 G2/M-M-G1 252 Bstt3-31-215 unknownproteinG2/M-M-G1 253 Bstt3-31-330 unknown G1/S-S-G2 254 Bstt33-1-350 unknownG2/M-M-G1 255 Bstt33-1-510 putativeprotein G2/M-M-G1 256 Bstt33-3-220unknown G2/M-M-G1 257 Bstt33-3-245 unknownprotein G2/M-M-G1 258Bstt3-33-550 hypotheticalprotein G1/S-S; M-G1 259 Bstt33-4-140putativeprotein S-G2 260 Bstt34-2-165 unknown G1/S-S-G2 261 Bstt3-42-325hypotheticalprotein G2/M-M-G1 262 Bstt3-44-150 unknown G2/M-M-G1 263Bstt3-44-250 unknown G2/M-M-G1 264 Bstt34-4-310 unknown G2/M-M-G1 265Bstt3-44-345 hypotheticalprotein G2/M-M-G1 266 Bstt41-2-340 G2/M-M-G1267 Bstt41-3-310 unknown G2/M-M 268 Bstt4-21-185 M-G1 269 Bstt42-1-370S-G2-G2/M 270 Bstt4-23-480 unknown G2/M-M-G1 271 Bstt4-24-170 G2/M-M-G1272 Bstt43-265 unknown G1/S-S-G2/M 273 Bstt43-3-350 unknown G2/M-M-G1274 Bstt4-33-390 hypotheticalprotein G1/S-S; G2/M- M-G1 275 Bstt4-34-280G2/M-M-G1 276 Bstt43-4-300 unknownprotein G2/M-M-G1 277 Bstt43-4-330unknownprotein G2/M-M-G1 278 Bstt43-4-340 G2/M-M-G1 279 Bstt44-4-250hypotheticalprotein G2/M-M 280 Bstt4-44-400 hypotheticalproteinG2/M-M-G1 281 MBc03-90 unknown S-G2 282 MBc42-180 unknown G2-M-G1 283MBc43-210 unknown G1/S-S-G2

TABLE 5 overview group 4 sequences showing no homology to known genesSEQ ID NO Tag name Function Fase 284 Bstc11-100 unknown G2/S-G2-M 285Bstc1-11-110 unknown S 286 Bstc1-11-115 unknown G1/S-S; G2/M-M-G1 287Bstc1-11-120 G1/S-S-G2 288 Bstc11-1-125 unknown G2/M-M-G1 289Bstc11-1-290 NaD G1/S; G2/M-M-G1 290 Bstc1-12-155 G2/S-G2-M 291Bstc1-12-175 unknown S 292 Bstc1-12-185 unknown G2/M-M-G1 293Bstc11-3-116 unknown S-G2 294 Bstc11-3-118 unknown G2/M-M-G1 295Bstc1-13-120 S 296 Bstc1-13-130 G1/S-S; G2/M-M-G1 297 Bsct1-13-132unknown M-G1 298 Bsct1-13-142 unknown G1/S-S 299 Bstc11-3-187 unknownS-G2/S 300 Bsct11-3-200 unknown G1/S-S-G2/S 301 Bsct11-3-290 unknownG2/S-G2-M-G1 302 Bsct1-14-100 unknown G2/M-M 303 Bstc1-14-108 unknownG2/M-M-G1 304 Bstc11-4-130 unknown G1/S-S-G2 305 Bsct11-4-135 unknownG2/M-M-G1 306 Bsct11-4-140 unknown S-G2-M 307 Bsct1-14-155 G2/M-M 308Bsct1-14-165 G2-G2/M 309 Bsct1-14-167 G2-G2/M 310 Bsct11-4-175 G2/M-M-G1311 Bsct11-4-200 unknown G1/S-S 312 Bstc12-1-110 unknown S-G2 313Bstc1-21-150 unknown G2/M-M-G1 314 Bstc12-1-160 unknown G2-M-G1 315Bstc12-1-240 unknown M-G1 316 Bstc12-1-95 unknown G1/S-S-G2 317Bstc1-22-110 G2-M-G1 318 Bstc12-3-103 unknown G2/M-M-G1 319 Bstc12-3-125unknown G1/S-S; G1 320 Bstc12-3-235 M-G1 321 Bstc12-3-237 unknown G1/S-S322 Bstc12-4-130 unknown G2/M-M-G1 323 Bstc12-4-133 unknown S-G2 324Bstc12-4-145 unknown M-G1 325 Bstc12-4-235 unknown G2/M-M-G1 326Bstc13-1-150 M-G1 327 Bstc13-2-170 unknown G2/M-M-G1 328 Bstc13-2-180unknown G1/S-S 329 Bstc13-2-190 unknown G1/S-S 330 Bstc13-2-280 unknownG1/S-S; G2/M-M-G1 331 Bstc1-41-170 unknown G1/S-S 332 Bstc1-41-175unknown G1/S-S 333 Bstc1-41-180 unknown G1/S-S; G2/M-M-G1 334Bstc1-41-210 unknown G1/S-S 335 Bstc1-41-230 G1/S; G2/M-M-G1 336Bstc14-2-140 unknown M-G1 337 Bstc1-42-150 unknown G2/S-G2 338Bstc1-42-80 unknown G1/S-S-G2 339 Bstc1-42-90 unknown G2-M 340Bstc1-43-105 G2/M-M 341 Bstc14-3-105 G1/S-S; G2/M-M 342 Bstc1-43-110G1/S-S; G2-M 343 Bstc14-3-130 unknown G2/M-M-G1 344 Bstc1-43-140 unknownS-G2 345 Bstc1-43-150 G2/M-M-G1 346 Bstc1-43-175 S-G2 347 Bstc1-43-185unknown G1/S-S-G2/S 348 Bstc14-3-235 unknown G1/S-S 349 Bstc14-3-260unknown G2/M-M-G1 350 Bstc1-43-65 unknown G1/S-S-G2 351 Bstc1-43-75unknown S-G2 352 Bstc1-44-138 unknown G1/S-S-G2/S 353 Bstc1-44-140unknown G2/S-G2-M 354 Bstc1-44-157 unknown G2/S-G2 355 Bstc14-95 unknownG2/M-M 356 Bstc21-1-100 unknown G2/M-M-G1 357 Bstc21-1-140 unknownG1/S-S-G2 358 Bstc21-1-145 unknown M-G1 359 Bstc21-1-65 unknown G2-M-G1360 Bstc21-2-120 G2/M-M 361 Bstc21-2-215 G2/M-M 362 Bstc21-2-75 S-G2-M363 Bstc2-13-110 G1/S-S; G2/M-M 364 Bstc2-14-100 unknown G2/M-M-G1 365Bstc21-4-120 unknown M-G1 366 Bstc2-14-125 unknown G2/M-M-G1 367Bstc21-4-130 unknown G2/M-M-G1 368 Bstc2-14-135 unknown S-G2/S 369Bstc21-4-135 S-G2 370 Bstc21-4-155 unknown G2/M-M-G1 371 Bstc2-14-160M-G1 372 Bstc21-4-180 unknown G2/S-G2 373 Bstc22-100 unknown G2-M 374Bstc2-21-120 unknown G1/S-S 375 Bstc22-1-125 unknown S-G2 376Bstc2-21-170 unknown M-G1 377 Bstc22-1-98 unknown S-G2-G2/M 378Bstc22-2-110 unknown G2/M-M-G1 379 Bstc2-22-160 unknown G1/S-S; G2-G2/M380 Bstc22-2-165 unknown G1/S-S 381 Bstc2-22-90 S; G2-M 382 Bstc2-23-110unknown G2/M-M 383 Bstc2-23-140 M-G1 384 Bstc22-3-150 S-G2 385Bstc2-23-175 M-G1 386 Bstc2-23-195 unknown M-G1 387 Bstc22-3-90 M-G1 388Bstc2-24-100 unknown G2/M-M-G1 389 Bstc22-4-140 G1/S-S-G2-M 390Bstc2-24-165 G2/M-M 391 Bstc2-24-170 unknown G1/S-S 392 Bstc2-31-140unknown G2/M-M-G1 393 Bstc2-31-160 M-G1 394 Bstc2-31-170 unknown M-G1395 Bstc23-2-135 unknown G2/M-M-G1 396 Bstc2-32-285 G2/M-M 397Bstc23-2-360 unknown G1/S; G2/M-M-G1 398 Bstc23-2-80 unknown G2/M-M 399Bstc23-3-175 unknown G1/S-S-G2 400 Bstc2-33-200 unknown G2/M-M-G1 401Bstc23-3-305 unknown M-G1 402 Bstc2-33-85 S-G2 403 Bstc2-33-95 unknownG2/M-M-G1 404 Bstc23-4-110 unknown G2-M 405 Bstc23-4-120 unknownG1/S-S-G2 406 Bstc23-4-310 S-G2 407 Bstc23-4-335 G2-M-G1 408Bstc2-41-110 unknown S-G2 409 Bstc24-2-165 M-G1 410 Bstc2-43-105 unknownS-G2-G2/M 411 Bstc2-43-130 unknown G2/M-M 412 Bstc24-3-285 G1 413Bstc2-43-77 unknown G2/M-M-G1 414 Bstc2-43-90 unknown G2/M-M-G1 415Bstc24-4-125 unknown G1/S-S 416 Bstc2-44-175 unknown G2/M-M-G1 417Bstc24-4-220 G2/M-M-G1 418 Bstc24-4-230 G2-G2/M 419 Bstc2-44-95 unknownM-G1 420 Bstc31-110 unknown G1/S-S 421 Bstc31-1-250 G2/M-M 422Bstc31-1-77 M-G1 423 Bstc31-1-90 unknown M-G1 424 Bstc3-12-115 unknownM-G1 425 Bstc31-2-190 unknown G1/S-S-G2 426 Bstc31-3-127 unknownG1/S-S-G2/M 427 Bstc31-3-235 unknown S-G2 428 Bstc3-13-330 G1 429Bstc31-3-60 unknown G2-M 430 Bstc31-3-80 unknown S-G2-M-G1 431Bstc3-13-90 unknown G2/M-M-G1 432 Bstc3-13-95 unknown M-G1 433Bstc3-14-105 unknown M-G1 434 Bstc3-14-110 unknown M-G1 435 Bstc3-14-125unknown G2/M-M-G1 436 Bstc3-14-130 unknown G1/S; M-G1 437 Bstc32-1-108unknown G1/S-S-G2 438 Bstc32-1-170 unknown S-G2/S 439 Bstc3-21-70unknown M-G1 440 Bstc32-2-100 unknown G1/S-S-G2 441 Bstc32-2-270 unknownG1/S; G2/M-M-G1 442 Bstc32-2-390 unknown G2/M-M-G1 443 Bstc32-2-93unknown G2/M-M 444 Bstc32-3-100 unknown S-G2 445 Bstc3-23-125 unknownG2/M-M-G1 446 Bstc32-3-155 S-G2-M 447 Bstc3-23-175 unknown G2/M-M-G1 448Bstc3-23-177 G2/S-G2-M-G1 449 Bstc32-3-63 unknown S-G2 450 Bstc3-23-65S; G2-M-G1 451 Bstc3-24-155 unknown G2/M-M-G1 452 Bstc32-4-230 unknownG2/M-M 453 Bstc32-4-250 unknown G2/M-M-G1 454 Bstc3-24-255 unknownG2/M-M-G1 455 Bstc3-24-305 G2-M-G1 456 Bstc3-24-340 unknown G1/S-S; M-G1457 Bstc3-24-90 M-G1 458 Bstc3-31-130 unknown G1/S-S-G2 459 Bstc33-120unknown G1/S-S 460 Bstc3-31-200 S-G2 461 Bstc3-31-260 unknown G1/S-S 462Bstc33-150 unknown G2/M-M-G1 463 Bstc3-32-105 unknown G2-G2/M 464Bstc3-32-120 G1/S-S; G2/M-M-G1 465 Bstc3-32-240 unknown S-G2 466Bstc3-32-320 G1/S-S-G2; M-G1 467 Bstc33-280 unknown G2-M-G1 468Bstc33-2-90 unknown S-G2 469 Bstc33-3-105 unknown G2/M-M-G1 470Bstc33-3-115 G1/S-S; M-G1 471 Bstc33-3-165 G1/S-S-G2/S 472 Bstc3-34-110G2/M-M 473 Bstc33-4-165 G2/M-M 474 Bstc33-4-200 S 475 Bstc3-34-290unknown G2/M-M-G1 476 Bstc3-34-85 unknown G2-M-G1 477 Bstc3-34-90unknown G1/S-S 478 Bstc33-90 unknown S 479 Bstc34-115 G2-M-G1 480Bstc3-41-180 G2/M-M-G1 481 Bstc34-13-300 unknown G/S-S; M-G1 482Bstc34-3-100 M-G1 483 Bstc34-3-135 S-G2-G2/M 484 Bstc34-3-190 S-G2-M-G1485 Bstc3-43-210 unknown G1/S-S; M-G1 486 Bstc34-3-210 unknownG2/S-G2-G2-G2/M 487 Bstc3-43-240 G1/S-S; G2/M-M-G1 488 Bstc34-3-248unknown S 489 Bstc34-3-263 unknown G2/M-M-G1 490 Bstc3-43-280 unknownG2/M-M-G1 491 Bstc34-3-95 unknown S 492 Bstc3-44-155 unknown G1/S-S;M-G1 493 Bstc3-44-173 G2/M-M-G1 494 Bstc34-80 unknown S-G2/S 495Bstc4-11-117 G2/M-M-G1 496 Bstc41-1-125 unknown M-G1 497 Bstc41-1-130unknown G2-M-G1 498 Bstc4-11-180 G2/M-M-G1 499 Bstc41-1-195 unknownG1/S-S-G2 500 Bstc41-1-197 unknown G2/M-M-G1 501 Bstc4-11-210 unknownG1/S-S-G2/S 502 Bstc41-1-210 unknown G1/S-S-G1/S 503 Bstc41-1-245unknown M-G1 504 Bstc4-11-350 unknown G2/M-M 505 Bstc41-1-90 unknownG2/M-M-G1 506 Bstc4-12-150 unknown G2-M-G1 507 Bstc41-2-280 S-G2-M 508Bstc4-13-112 unknown S-G2 509 Bstc41-3-170 unknown G1/S-S 510Bstc41-3-205 unknown G2/M-M-G1 511 Bstc4-13-280 unknown G1/S-S-G2/S 512Bstc4-13-70 unknown G2/M-M-G1 513 Bstc41-4-105 M-G1 514 Bstc41-4-112unknown G2/M-M 515 Bstc4-14-120 unknown G1/S-S; M-G1 516 Bstc41-4-127unknown S-G2-M 517 Bstc41-4-145 unknown G2/M-M-G1 518 Bstc4-14-160unknown G2/M-M-G1 519 Bstc41-4-165 unknown G2-M-G1 520 Bstc41-4-185G1/S-S-G2 521 Bstc41-4-270 G1/S-S; G2/M-M-G1 522 Bstc42-1-150 unknownG2/M-M-G1 523 Bstc4-21-155 G1/S-S-G2 524 Bstc4-21-200 unknown S;G2/M-M-G1 525 Bstc42-135 unknown G2/M-M-G1 526 Bstc4-22-150 unknownG1/S-S; G1 527 Bstc42-2-170 S-G2-M 528 Bstc42-2-185 M-G1 529Bstc42-2-220 unknown M-G1 530 Bstc42-3-100 unknown M-G1 531 Bstc4-23-115unknown M-G1 532 Bstc42-3-133 S-G2/S 533 Bstc4-23-135 unknown G2/M-M-G1534 Bstc42-4-110 unknown G1/S-S; G2/M-M-G1 535 Bstc4-24-240 G1/S-S-G2536 Bstc4-31-260 G2/M-M-G1 537 Bstc4-31-310 unknown S; G2/M-M-G1 538Bstc43-3-100 S-G2-M 539 Bstc43-3-103 unknown G2/M-M-G1 540 Bstc43-3-135M-G1 541 Bstc43-3-175 G2/M-M-G1 542 Bstc43-3-250 unknown M-G1 543Bstc4-34-135 unknown G2/M-M-G1 544 Bstc4-34-185 G1/S-S 545 Bstc43-4-200unknown G2/M-M-G1 546 Bstc43-4-320 G1/S-S 547 Bstc4-41-100 unknown G2-M548 Bstc4-41-105 unknown G1/S-S; G2/M-M-G1 549 Bstc4-41-107 unknownG2/M-M-G1 550 Bstc4-41-125 unknown M-G1 551 Bstc4-41-180 G2/M-M-G1 552Bstc4-41-220 unknown M-G1 553 Bstc44-150 unknown G2-M-G1 554Bstc4-42-110 unknown G1/M-M-G1 555 Bstc4-42-115 unknown G2/M-M 556Bstc4-42-130 unknown S-G2 557 Bstc4-42-165 unknown G1/S-S; M-G1 558Bstc4-42-217 unknown G2/M-M-G1 559 Bstc4-43-103 unknown G1/S-S-G2-G2/M560 Bstc44-3-167 unknown G2/M-M-G1 561 Bstc44-3-170 M-G1 562Bstc44-4-120 unknown M-G1 563 Bstc44-4-290 unknown G2/M-M-G1 564Bstt1-11-190 G1/S-S 565 Bstt1-11-200 unknown G1/S-S-G2-G2/M 566Bstt1-11-55 unknown G1/S-S 567 Bstt1-11-65 unknown G1/S-S-G2 568Bstt1-12-105 unknown G2/M-M 569 Bstt1-12-115 G1/S-S 570 Bstt1-12-230S-G2 571 Bstt1-13-150 unknown G2/M-M 572 Bstt1-13-230 unknown G2/S-G2-M573 Bstt1-14-125 unknown G1/S-S 574 Bstt1-14-220 unknown G2/M-M 575Bstt1-21-100 unknown G2/M-M 576 Bstt12-1-240 unknown S-G2-M 577Bstt1-21-250 unknown S; G2/M-M-G1 578 Bstt12-2-100 unknown G2/S-G2-M-G1579 Bstt12-2-140 unknown G2/M-M-G1 580 Bstt1-22-160 G2/M-G1 581Bstt12-2-215 unknown G2/M-M 582 Bstt1-22-225 M-G1-G1/S 583 Bstt12-2-360unknown G2/M-M-G1 584 Bstt1-22-70 unknown G1/S-S 585 Bstt12-3-115unknown G1/S-S-G2 586 Bstt1-23-150 unknown G2-M-G1 587 Bstt1-23-170unknown G2-M 588 Bstt12-3-170 unknown G1/S-S 589 Bstt1-23-180 unknownG2/S-G2-M 590 Bstt1-23-185 G2-M-G1 591 Bstt1-23-235 unknown G2-M 592Bstt1-24-105 unknown G2/S-G2-M-G1 593 Bstt1-24-120 unknown G2/M-M-G1 594Bstt12-4-260 G2/S-G2-G2/M 595 Bstt12-4-320 G2/M-M 596 Bstt1-31-120G2/M-M-G1 597 Bstt1-31-180 unknown G2/M-M-G1 598 Bstt13-170 unknownG1/S-S-G2 599 Bstt13-2-150 G1/S-S-G2 600 Bstt1-32-170 unknown G1/S-S-G2601 Bstt1-32-185 G1/S-S 602 Bstt13-3-100 unknown G1/S-S-G2-M 603Bstt1-33-170 unknown G1/S-S-G2 604 Bstt13-3-320 unknown G2/M-M-G1 605Bstt1-33-66 G2/M-M 606 Bstt1-41-120 unknown G2/M-M 607 Bstt1-42-264unknown G2-M-G1 608 Bstt14-2-280 unknown G2/M-M-G1 609 Bstt14-3-120 S-G2610 Bstt14-3-140 unknown G1-S-S-G2 611 Bstt1-43-220 unknown G2/S-G2-G2/M612 Bstt1-43-330 unknown G2/M-M-G1 613 Bstt14-3-460 unknown G2/M-M 614Bstt14-4-130 unknown S-G2 615 Bstt14-4-150 unknown G2 616 Bstt14-4-195S-G2-M 617 Bstt14-4-220 G2/S-G2-G2/M 618 Bstt14-85 nohits G2/M-M 619Bstt21-1-170 unknown G2/M-M 620 Bstt2-11-290 G2/S-G2-G2/M 621Bstt2-11-540 G1/S-S 622 Bstt21-2-190 G2/M-M-G1 623 Bstt2-13-165 S-G2-M624 Bstt2-13-170 unknown G2/M-M 625 Bstt2-14-130 unknown G2/M-M 626Bstt2-14-175 unknown S-G2 627 Bstt22-1-140 unknown S-G2 628 Bstt2-21-300unknown G2/M-M 629 Bstt22-2-110 unknown G1/S-G2 630 Bstt22-2-255G1/S-S-G2-G2/M 631 Bstt22-2-370 G1/S-G2 632 Bstt22-3-100 unknownG2/M-M-G1 633 Bstt22-3-145 unknown G2/M-M-G1 634 Bstt2-23-220 unknownG2-M-G1 635 Bstt2-23-370 G1/S-G2 636 Bstt22-4-145 unknown G2/M-M 637Bstt22-4-170 S-G2 638 Bstt22-4-175 G2-M 639 Bstt22-80 unknown G2/M-M 640Bstt23-1-128 unknown S-G2 641 Bstt23-1-155 unknown S-G2-G2/M 642Bstt2-31-200 unknown G2/S-G2 643 Bstt23-170 unknown G2/M-M-G1 644Bstt2-32-175 unknown G2/S-G2-G2/M 645 Bstt23-220 G1/S-S-G2 646Bstt23-3-200 G1/S-S-G2/S 647 Bstt23-3-265 S-G2-G2/M 648 Bstt23-3-330G1/S-S 649 Bstt2-34-170 unknown G2/M-M-G1 650 Bstt23-4-180 S-G2-M 651Bstt23-4-210 G2/M-M-G1 652 Bstt2-41-170 unknown G1/S-S-G2 653Bstt24-1-170 unknown S-G2 654 Bstt2-41-390 S-G2 655 Bstt2-42-300G2/M-M-G1 656 Bstt24-2-318 S-G2 657 Bstt24-2-320 unknown G2/M-M-G1 658Bstt24-290 unknown G2/M-M 659 Bstt2-43-150 S-G2 660 Bstt2-43-160 S-G2/S661 Bstt2-43-50 S 662 Bstt2-43-65 unknown S-G2 663 Bstt2-44-230G2/S-G2-M 664 Bstt2-44-240 unknown G1/S-S-G2 665 Bstt24-4-240 unknownG1/S-S-G2/S 666 Bstt24-4-260 unknown G1/S-S 667 Bstt24-4-283 unknownG1/S-S-G2 668 Bstt24-4-285 unknown G2/M-M-G1 669 Bstt31-1-145 S-G2-M 670Bstt31-1-210 G2/M-M-G1 671 Bstt31-2-165 unknown G2/S-G2 672 Bstt31-2-185G2/M-M-G1 673 Bstt3-12-200 unknown G2/M-M-G1 674 Bstt3-12-315 S-G2-M 675Bstt31-2-330 G2/M-M-G1 676 Bstt3-13-110 unknown S-G2-G2/M 677Bstt31-3-180 S-G2-G2/M 678 Bstt3-13-360 G2/M-M 679 Bstt3-14-130 unknownG2/M-M 680 Bstt3-14-135 unknown G2/M-M 681 Bstt31-50 unknownG1/S-S-G2-G2/M 682 Bstt32-1-105 S-G2 683 Bstt3-21-165 G2/S-G2 684Bstt3-21-305 unknown G2/M-M 685 Bstt32-140 unknown S-G2/S 686Bstt3-22-100 G2/M-M-G1 687 Bstt32-2-210 S-G2-M 688 Bstt3-22-280 unknownG1/S-S; M-G1 689 Bstt32-2-510 unknown S-G2-G2/M 690 Bstt32-3-115 G2/S-G2691 Bstt32-3-155 unknown S-G2 692 Bstt32-3-160 M 693 Bstt32-3-180unknown G1/S-S-G2 694 Bstt3-23-205 unknown S-G2-M 695 Bstt3-23-65unknown G2/M-M-G1 696 Bstt32-4-170 unknown S; M 697 Bstt32-4-195 G1/S-S;G2/M-M-G1 698 Bstt32-4-260 unknown G1/S-S 699 Bstt3-24-390 M-G1 700Bstt33-1-105 G1/S-S-G2 701 Bstt33-1-128 S-G2 702 Bstt33-1-132 unknownG2/M-M 703 Bstt33-1-160 unknown G2/M-M-G1 704 Bstt33-1-185 M-G1 705Bstt33-140 unknown G2/M-M-G1 706 Bstt33-2-75 unknown G1/S-S-G2 707Bstt33-2-85 G1/S-S; G2/M-G1 708 Bstt33-3-110 G1/S-S; G2/M-M-G1 709Bstt33-3-125 unknown G2/M-M-G1 710 Bstt3-33-170 unknown S-G2/S 711Bstt33-4-110 S-G2 712 Bstt33-4-120 unknown G1/S-S-G2 713 Bstt33-4-130unknown G2/M-M 714 Bstt33-95 unknown G2/M-M 715 Bstt34-1-110 S-G2-G2/M716 Bstt34-1-170 G1/S-S-G2-G2/M 717 Bstt3-42-350 unknown G2/M-M-G1 718Bstt3-43-145 unknown G2/M-M-G1 719 Bstt3-43-190 unknown G1/S-S; M-G1 720Bstt3-43-265 G2/S-G2-M-G1 721 Bstt3-43-280 unknown G2/M-M-G1 722Bstt34-70 unknown S 723 Bstt41-3-100b unknown 2/M-M 724 Bstt41-3-130unknown G2/M-M-G1 725 Bstt41-3-140 unknown G2/M-M-G1 726 Bstt41-3-180G2-M 727 Bstt41-3-230 unknown S-G2 728 Bstt41-3-90 unknown G2/M-M-G1 729Bstt41-4-210 unknown S-G2-M-G1 730 Bstt4-14-500 G2/M-M-G1 731 Bstt41-70unknown G1/S-S 732 Bstt42-1-130 unknown G2/M-M-G1 733 Bstt42-1-290unknown G2/M-M 734 Bstt4-21-60 unknown S-G2 735 Bstt4-22-100 M-G1 736Bstt4-22-360 S-G2 737 Bstt42-3-105 unknown G1/S-S-G2/S 738 Bstt42-3-110unknown G2/M-M-G1 739 Bstt4-23-130 S-G2/M 740 Bstt4-23-160 G2/S-G2-M 741Bstt42-4-150 unknown G1/S-S-G2 742 Bstt4-24-270 unknown G2/M-M-G1 743Bstt42-4-390 unknown M-G1 744 Bstt43-1-290 unknown G2/M-M-G1 745Bstt43-1-85 G1/S-S-G2/S 746 Bstt4-32-230 unknown G1/S-S-G2/S 747Bstt43-2-238 G2/M 748 Bstt43-3-145 unknown G1/S-S-G2 749 Bstt43-3-210G2/M-M-G1 750 Bstt43-4-230 unknown G2/M-M-G1 751 Bstt4-34-75 unknownG2/S-G2-M 752 Bstt44-1-125 unknown S-G2-G2/M 753 Bstt44-185 unknown M-G1754 Bstt44-2-135 G2/M-M-G1 755 Bstt4-42-150 unknown M 756 Bstt4-42-390unknown M-G1 757 Bstt44-3-240 unknown G2/M-M-G1 758 Bstt44-3-250 unknownS-G2-G2/M 759 Bstt4-44-148 G2/M-M-G1 760 Bc02-100 unknown G2/M-M 761Bc02-120 unknown G2/M-M 762 Bc03-110 unknown G2/M-M 763 Bc03-85 G2/M-M764 Bc11-135 unknown G2-M 765 Bc12-150 S-G2-M 766 Bc31-185 unknownG2/M-M 767 M Bc32-107 unknown G2/M-M-G1 768 M Bc32-110 unknown G2/M-M-G1769 M Bc41-110 unknown G1/S-S; G2/M-M 770 M Bc42-280 unknown G2-M 771 MBc43-95 unknown G2-M 772 M Bc44-130 S-G2 773 M Bc44-95 unknown G2/M-M774 M Bt12-80 unknown G2/M-M 775 M Bt12-95 M 776 M Bt13-105 unknown M-G1777 M Bt14-100 unknown G2/M-M-G1 778 M Bt14-85 unknown S-G2-M 779 MBt14-90 unknown G2-M 780 M Bt31-95 S-G2-M 781 M Bt33-115 G2/M-M-G1 782 MBt33-133 G2-M 783 M Bt42-135 unknown G2-M 784 M Bt43-95 unknown G2-G2/M785 M Bt44-145 unknown G1/S-S-G2-M

1-16. (canceled) 17: A method for identifying and validating plantgenes/proteins as targets for agrochemicals, said method comprising thesteps of: a. determining gene or protein expression profiles during abiological process of a plant or plant cell, said biological processbeing necessary for the growth and/or development and/or viability ofthe plant or plant cell; b. selecting genes or proteins having alteredexpression during said biological process; c. cloning said selected geneor the nucleic acid encoding said protein in its full-length or partialform; d. incorporating said nucleic acid in a vector designed fordownregulation of expression of said nucleic acid or the sequencehomologous to said nucleic acid in a plant or plant cell. 18: The methodaccording to claim 17, wherein said biological process is cell division.19: The method according to claim 17, wherein said gene or proteinexpression profiling is based on nucleic acid or protein samplescollected from a synchronized culture of dividing plant cells. 20: Themethod according to claim 19, wherein said dividing plant cells aretobacco BY2 cells. 21: The method according to claim 20, wherein theexpression profiles are determined by means of micro-array, macro arrayor c-DNA-AFLP. 22: The method according to claim 17, wherein saiddownregulation involves a viral-induced gene silencing mechanism. 23:The method according to claim 22, wherein said downregulation involvesthe use of infectious DNA of virus is Tobacco Rattle Virus and whereinsaid plant is tobacco. 24: The method for screening candidateagrochemical compounds comprising the method of claim
 17. 25: Anisolated nucleic acid identified by the method of claim
 17. 26: A methodfor screening candidate agrochemical compounds comprising using at leastone or more of SEQ ID NO 1 to 785 or a homologue, functional fragment orderivative thereof or at least one or more of the proteins correspondingto SEQ ID NO 1 to 785 or a homologue, functional fragment or derivativethereof. 27: An isolated nucleic acid identified by the method of claim26. 28: A method for the production of an agrochemical resistant plant,comprising using at least one or more of SEQ ID NO 1 to 785 or ahomologue, functional fragment or derivative thereof or at least one ormore of the proteins encoded by SEQ ID NO 1 to 785 or a homologue,functional fragment or derivative thereof. 29: An isolated nucleic acididentified by the method of claim
 28. 30: An isolated nucleic acid,comprising at least part of a nucleic acid sequence chosen from thegroup of SEQ ID NO 1 to 785 or a homologue, functional fragment orderivative thereof. 31: A method for producing an agrochemical resistantplant, comprising using the isolated nucleic acid or the protein encodedby said isolated nucleic acid of claim 29, wherein said isolated nucleicacid or protein is a target for an agrochemical compound. 32: The methodaccording to claim 31, wherein the agrochemical compound is a herbicide.33: A plant tolerant to an agrochemical, in which the expression levelof one or more of the nucleic acids corresponding to SEQ ID NO 1 to 785or the homologue, functional fragment or derivative thereof ismodulated. 34: A harvestable part of a plant according to claim 33.